Car and car-nk cells targeting bcma and gprc5d and their use in the treatment of multiple myeloma

Abstract

This document provides chimeric antigen receptors (CARs) and CAR-NK cells targeting BCMA and GPRC5D, and their use in the treatment of multiple myeloma (MM). Specifically, this disclosure relates to bispecific CARs, the corresponding encoding polynucleotides of the CARs, and vectors containing the polynucleotides, said CARs comprising: (a) a B cell maturation antigen (BCMA) targeting domain; and (b) a G protein-coupled receptor C family 5 member D (GPRC5D) targeting domain, preferably armed with IL-15, etc. Furthermore, this document also provides CAR-NK cells modified with one or more CARs, and the use of CARs and CAR-NK cells in the treatment of MM.

Description

Technical Field

[0001] This disclosure relates to the fields of genetic engineering and immunotherapy. In particular, it relates to genetically modified CAR-NK cells targeting BCMA and GPRC5D and their use in the treatment of multiple myeloma (MM). Background Technology

[0002] Multiple myeloma (MM) is a blood disorder that accounts for 10% of all hematologic malignancies and is the second most common blood cancer. MM is characterized by the abnormal proliferation of malignant plasma cells. Treatment modalities for MM have changed dramatically over the past few decades. However, even with new treatment strategies, most MM patients remain incurable and typically progress to refractory and drug-resistant multiple myeloma (RRMM).

[0003] In recent years, CAR-T (chimeric antigen receptor T-cell) cell therapy has brought new hope to patients with multiple myeloma (MM), especially those with recurrent partial myeloma (RRMM). These T cells are typically transduced using lentiviral or retroviral vectors carrying the CAR-encoding gene, then expanded in vitro, and finally infused into the patient. After infusion, these CAR-T cells release cytokines upon encountering antigens, lyse target cells, and proliferate in vivo.

[0004] B-cell maturation antigen (BCMA) is a type III transmembrane receptor that is an ideal target for immunotherapy because it is expressed almost exclusively in plasma cells and most plasma cell tumors. It is also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17) or CD269. Despite the encouraging clinical results of BCMA CAR-T therapy, most MM patients still face disease relapse after treatment. One possible reason for relapse is the heterogeneity of BCMA expression, which leads to resistance to BCMA CAR-T therapy in MM cells with low BCMA expression. Therefore, the search for new targets to combat the heterogeneity of MM cells remains.

[0005] GPRC5D is an orphan G protein-coupled receptor (PGR) belonging to group 5D of the C family and is a promising novel target for immunotherapy of multiple myeloma (MM). GPRC5D is highly and selectively expressed in MM cells, while in normal tissues, it is expressed only in cells that produce keratin (e.g., hair follicle cells). Furthermore, studies have found that the expression profile of GPRC5D in MM patients is independent of that of BCMA.

[0006] To date, CAR-T cell therapies have all been autologous products, meaning that the patient's own T cells are harvested, inserted into a CAR in the laboratory, and then reinfused. While the clinical efficacy of these autologous drugs in clinical studies is undeniable, their limitations in real-world application cannot be ignored, including high costs, restrictions on patient eligibility and / or performance status, risk of disease progression, long waiting times, high incidence of cytokine release syndrome (CRS), and manufacturing process issues. Therefore, the demand for allogeneic and off-the-shelf cell therapies is growing.

[0007] Due to their unique biological characteristics and multiple mechanisms of action, natural killer (NK) cells have emerged as a potent candidate therapy. Unlike T cells, adoptive NK or CAR-NK therapies do not cause severe cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). Allogeneic CAR-NK products overcome the expensive and time-consuming steps of preparing autologous CAR-T cells and, due to their extremely low risk of inducing graft-versus-host disease (GvHD), can be offered as an "off-the-shelf" immunotherapy product suitable for diverse patients and available from multiple sources. Furthermore, even in cases of tumor escape characterized by CAR antigen loss or downregulation, NK cells can maintain CAR-independent killing capacity through their intrinsic receptors, thereby reducing the risk of relapse.

[0008] For multiple myeloma (MM), a disease that can be highly heterogeneous, relapsed, or refractory, there remains an urgent need to develop new products and methods to effectively treat the disease. Summary of the Invention

[0009] This article discloses CARs targeting BCMA and GPRC5D and genetically modified CAR-NK cells and their use in the treatment of MM.

[0010] Based on several aspects, this article discloses a bispecific chimeric antigen receptor (CAR) comprising: (a) B cell maturation antigen (BCMA) targeting domain; and (b) Targeting domain of G protein-coupled receptor C family 5 member D (GPRC5D).

[0011] In some embodiments, the two target domains of the CAR are independently selected or derived from / from an antibody or its antigen-binding fragment. In some embodiments, the two target domains of the CAR are independently selected or derived from / from scFv, VHH, and nanobodies. In some embodiments, the two target domains of the CAR are tandemly linked, fused, or coupled to each other in any order, optionally with or without a linker.

[0012] In some implementations, the CAR is further armored to improve its performance, such as enhancing the cytotoxicity and persistence of the CAR or CAR-NK cells, and / or reducing cytokine release syndrome (CRS) of the CAR or CAR-NK cells.

[0013] In some implementations, the CAR is further armored with IL-15 or similar materials.

[0014] In some implementations, in addition to the target domain, the CAR also includes one or more of the following domains and / or units: (c) Extracellular signaling domains; (d) Extracellular hinge domain; (e) Transmembrane (TM) domain; (f) One or more intracellular signal transduction domains (ICDs); (g) Armored units; and (h) One or more independently selected joints between two domains or between a domain and a unit.

[0015] According to some aspects, this document discloses an anti-GPRC5D antibody or its antigen-binding fragment comprising a GPRC5D binding domain, wherein the GPRC5D binding domain includes heavy chain (VH) complementarity-determining regions (CDRs) 1, 2, and 3, and wherein VH CDR1 has the amino acid sequence SEQ ID NO: 25, VH CDR2 has the amino acid sequence SEQ ID NO: 26, and VH CDR3 has the amino acid sequence SEQ ID NO: 27. In some embodiments, the anti-GPRC5D antibody or its antigen-binding fragment is used to construct a CAR or NK cells modified with the CAR.

[0016] According to some aspects, this document discloses an anti-BCMA antibody or its antigen-binding fragment comprising a BCMA-binding domain, wherein the BCMA-binding domain includes heavy chain (VH) complementarity-determining regions (CDRs) 1, 2, and 3, and wherein VH CDR1 has the amino acid sequence SEQ ID NO: 16, VH CDR2 has the amino acid sequence SEQ ID NO: 17, and VH CDR3 has the amino acid sequence SEQ ID NO: 18; or wherein VH CDR1 has the amino acid sequence SEQ ID NO: 19, VH CDR2 has the amino acid sequence SEQ ID NO: 20, and VH CDR3 has the amino acid sequence SEQ ID NO: 21; or VH CDR1 has the amino acid sequence SEQ ID NO: 22, VH CDR2 has the amino acid sequence SEQ ID NO: 23, and VH CDR3 has the amino acid sequence SEQ ID NO: 24. In some embodiments, the anti-BCMA antibody or its antigen-binding fragment is used to construct a CAR or NK cells modified with the CAR.

[0017] According to several aspects, this document discloses one or more polynucleotide molecules encoding the CAR of this application.

[0018] Based on several aspects, this paper discloses one or more vectors containing CAR-encoding polynucleotide molecules.

[0019] According to some aspects, this document discloses modified NK cells that contain one or more CAR or polynucleotide molecules of this application, or are transduced using one or more vectors of this application.

[0020] According to some aspects, this document discloses products comprising one or more CARs, polynucleotide molecules, vectors, and CAR-NK cells.

[0021] According to some aspects, this document discloses a method for preparing the modified NK cells of this application, the method comprising the steps of: (i) modifying NK cells to contain the armored or unarmored CAR of this application; and (ii) optionally, expanding and collecting the modified NK cells containing the CAR.

[0022] According to several aspects, this document discloses the use of the modified NK cells of this application in the preparation of products for the treatment of multiple myeloma (MM).

[0023] According to several aspects, this article discloses a method for treating MM in subjects with this need, the method comprising administering an effective amount of the modified NK cells of this application.

[0024] In some respects, this article discloses a modified NK cell for the treatment of MM.

[0025] Other objects, features, advantages, and aspects of this application will become apparent to those skilled in the art from the following description and appended claims. However, it should be understood that while the following description, appended claims, and specific embodiments illustrate preferred embodiments of the application, they are given by way of illustrative purpose only. Various changes and modifications made within the spirit and scope of the disclosed invention will be apparent to those skilled in the art upon reading the following. Attached Figure Description

[0026] The appended claims specifically describe the novel features of the invention. The features and advantages of the invention can be better understood with reference to the following detailed description, which sets forth illustrative embodiments utilizing the principles of the invention and the accompanying drawings, wherein: Figure 1 The antibody against BCMAW3566 VHH-Fc (human IgG1) was shown to interact with BCMA on the cell surface. Figure 1 A, NCI-H929 cell line) and cynoBCMA ( Figure 1 B,293T overexpression of cynoBCMA binding.

[0027] Figure 2 The anti-GPRC5D antibody was shown to be effective against the human GPRC5D-positive cell line MM.1R ( Figure 2 A) Human GPRC5D positive cell line OPM-2 ( Figure 2 B) and GPRC5D negative cell line Nalm-6 ( Figure 2 The combination of C).

[0028] Figure 3 The binding of the anti-GPRC5D antibody to the cyno GPRC5D overexpressing cell line (293F-cyno GPRC5D) was demonstrated.

[0029] Figure 4 The CAR map of the retroviral plasmid vector pMSCV-BCMA-1D5xGPRC5D-1G4-sIL15 is shown. Figure 4 A) and the core components of CAR ( Figure 4 B).

[0030] Figure 5This study presents a representative CAR expression analysis on CAR-NK cells using BD FACS CantoII assays, including tandem leader CARs (BCMA-1D5xGPRC5D-1G4 tandem CAR), BCMA-1D5 monovalent CAR, BCMA-BMK CAR, GPRC5D-1G4 monovalent CAR, and GPRC5D-BMK CAR.

[0031] Figure 6 Various multiple myeloma cell lines were shown. Figure 6 A) or BCMA knockout (BCMA-KO) or GPRC5D knockout (GPRC5D KO) OPM2 cells ( Figure 6 B) Membrane expression of BCMA or GPRC5D proteins.

[0032] Figure 7 It shows CAR-NK versus OPM2 ( Figure 7 A), NCI-H929 ( Figure 7 B), RPMI-8226 ( Figure 7 C), BCMA-KOOPM2 ( Figure 7 D) and GPRC5D-KO OPM2 ( Figure 7 E) cytotoxicity.

[0033] The killing effect on target cells was determined by detecting green fluorescence using the Operetta CLS high-content analysis system (PerkinElmer) and was standardized relative to the killing effect of target tumor cells cultured alone (N=3, mean ± SEM).

[0034] Figure 8 The tumor eradication of CAR-NK cells in the OPM2 hybrid model (composed of 50% BCMA-KO OPM2-ZsGreen cells and 50% GPRC5D-KO OPM2-ZsGreen cells) is shown.

[0035] The green fluorescence signal of remaining live target cells was detected using the Incucyte S3 live cell analysis system (Sartorius). Compared with single CAR and BMK, the tandem lead (BCMA-1D5xGPRC5D-1G4) showed stronger tumor clearance in a mixed MM cell model (N=3, mean ± SEM).

[0036] Figure 9 It showed that CAR-NK cells interacted with target cells OPM2 ( Figure 9 A) or NCI-H929 ( Figure 9 B) IFN-γ secretion during co-culture.

[0037] CAR-NK cells and OPM2 ( Figure 9 A; co-culture for 2 days; initial target cell density is 4e5 cells / ml) or NCI-H929 ( Figure 9 B; co-cultured for 1 day; the initial density of fixed target cells was 4e5 cells / ml) co-cultured at a ratio of 1:1 or 0.5:1, and the supernatant was collected for IFN-γ detection (N=3, mean ± SEM).

[0038] Figure 10 The cytotoxicity of CAR-NK against target cells OPM2-ZsGreen was demonstrated: the IL15 armor gene enhanced the cytotoxicity of CAR-NK. Figure 10 A). Furthermore, the cytotoxicity of IL-15 armored CAR-NK cells is stronger than that of CAR-T cells. Figure 10 B).

[0039] Target cell killing was determined using the Operetta CLS high-content analysis system (PerkinElmer) to detect green fluorescence, and was standardized relative to the killing of target tumor cells cultured alone (n = 3, technical replication, two-way ANOVA to compare tandem lead + sIL15 (secreted IL-15) with tandem lead (A), tandem lead with control NK (A), and tandem lead + sIL-15 with tandem lead CAR-T (B). P < 0.0001, P < 0.001, P < 0.01). Data are expressed as mean ± SEM.

[0040] Figure 11 Sustained cytotoxicity of IL15-armored CAR-NK, CAR-NK without the IL15-armor gene, and CAR-T was demonstrated.

[0041] Target cells were added every 3 or 4 days to test the repeatable killing ability of CAR-NK and CAR-T cells. GFP signaling in remaining live target cells was detected using the Incucyte S3 live cell analysis system (Sartorius). IL15-armored CAR-NK cells exhibited the best sustained cytotoxic activity after 4 rounds of tumor cell attack (n = 3, technical replicates, two-way ANOVA, compared with tandem lead + sIL15). P < 0.0001). Data are expressed as mean ± SEM.

[0042] Figure 12 The study showed the secretion of IFN-γ when CAR-NK and CAR-T cells were co-cultured with target cells OPM2.

[0043] CAR-NK cells and OPM2 cells were co-cultured at a ratio of 1:1 or 0.5:1 (co-cultured for 2 days, with an initial target cell density of 4e5 cells / ml), and the supernatant was collected for IFN-γ detection. CAR-NK cells produced significantly less IFN-γ than CAR-T cells (n = 3, technical replication, two-way ANOVA compared tandem CAR-NK, tandem CAR-NK+ sIL15, and tandem CAR-T). P < 0.0001, P < 0.001, ns = no statistical significance), data are expressed as mean ± SEM.

[0044] Figure 13 Imaging was demonstrated using bioluminescence imaging. Figure 13 B) and drawing ( Figure 13 A) Presented tumor burden in mice injected with wild-type OPM2-luciferase cells, followed by treatment for 3 weeks with a single dose of IL15-armored BCMAxGPRC5D tandem CAR-NK, BCMAxGPRC5D CAR-NK (without IL15), NK-mimicking cells (CD19 CAR-NK with IL15 armor), or a carrier (200 μl cryopreservation buffer). (n = 4, technical replication, two-way ANOVA comparison: 1. Tandem CAR-NK + sIL15 vs. tandem CAR-NK (without IL15)) 2. Connect CAR-NK+sIL15 in series with simulated NK. 3. Connect CAR-NK (without IL15) and simulated NK in series. . P < 0.0001, P<0.001, P<0.05). Data are expressed as mean ± SEM. Detailed Implementation

[0045] The following description and examples illustrate embodiments of the present invention in detail. It should be understood that the present invention is not limited to the specific embodiments described herein and therefore can be modified. Those skilled in the art will recognize that the present invention has many variations and modifications, all of which are included within the scope of the present invention.

[0046] In this study, we introduced a tandem CAR targeting BCMA and GPRC5D into peripheral blood mononuclear cells (PBMCs) to prepare derived and expanded NK cells in vitro. These BCMA×GPRC5D tandem CAR-NK cells targeted and killed MM cells through the innate immune functions of BCMA, GPRC5D, and NK cells. Furthermore, to further enhance the proliferation, persistence, and cytotoxicity of CAR-NK cells, we also inserted the IL15 armor gene into the NK cells. The in vitro and in vivo efficacy of BCMAxGPRC5D tandem CAR-NK cells has been validated, providing a novel and promising treatment option for MM patients.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this document applies. While this disclosure may be practiced or tested using any methods and materials similar to or equivalent to those described herein, preferred methods and materials are described.

[0048] The terms “a” or “an” as used herein are intended to indicate the grammatical object of the article “one or more” (i.e., at least one). Unless otherwise defined in the context, singular expressions include plural expressions. For example, “one element” means one or more elements.

[0049] "About" or "approximately" means that the change in quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length can be 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0050] Unless otherwise stated, "or" means "and / or".

[0051] As used herein, unless otherwise stated, the terms “comprising,” “including,” and “containing” are to be understood as implying that the specified step or element or group of steps or elements is included, and do not exclude any other step or element or group of steps or elements.

[0052] The phrase “consisting of” is intended to include and be limited to anything that follows the phrase “consisting of”. Therefore, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that other elements may be present.

[0053] The term "isolated" refers to material that is substantially or essentially free from the components that normally accompany it in its natural state. This material can be cells or macromolecules such as proteins or nucleic acids. For example, as used herein, "isolated cell" refers to a cell purified from cells in their natural state.

[0054] As used herein, the term "antibody" encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, nanobody (VHH), multispecific antibody, or bispecific (bivalent) antibody that binds to one or more specific antigens. Typically, in the case of a naturally occurring intact antibody, an antibody consists of two heavy chains and two light chains. However, some specialized antibodies possess only a VH domain and lack a VL domain, yet remain remarkably stable; these are examples of antibodies called VHH antibodies or nanobodies.

[0055] Each heavy chain contains a variable region (“VH”) and first, second, and third constant regions (CH1, CH2, CH3), as well as a conditionally fourth constant region (CH4), as in the case of IgM and IgE antibodies, while each light chain consists of a variable region (“VL”) and a constant region (CL). Mammalian heavy chains are classified as α, δ, ε, γ, and μ, while mammalian light chains are classified as λ or κ. The variable regions of both light and heavy chains are responsible for antigen binding.

[0056] Each variable region typically contains three highly variable loops called “complementarity-determining regions (CDRs)”. As used herein, the term “CDR” or “complementarity-determining region” refers to a discontinuous antigen-binding site within the variable region of the heavy chain and / or light chain polypeptide. CDR boundaries can be defined or identified using conventional methods such as the Kabat, Chothia, IMGT, Al-Lazikani numbering schemes, or combinations thereof. The three CDRs are inserted between side-connecting sequences called frame regions (FRs), which are more conserved than the CDRs and form a scaffold supporting the highly variable loops. In some embodiments, one or more CDRs are identified according to the Kabat numbering scheme, the IMGT numbering scheme, or a combination of the IMGT and Kabat numbering schemes.

[0057] The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. The five major classes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are further divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). Therefore, in the context of this invention, a specific IgG isotype (e.g., "IgG1" or "IgG1 isotype") refers to the IgG isotype of the defined subclass, while different IgG isotypes refer to IgG isotypes of different subclasses.

[0058] The term "NK cell" or "natural killer cell" refers to a class of cytotoxic lymphocytes that are crucial to the innate immune system. NK cells mediate anti-tumor and antiviral responses, and therefore have broad clinical application prospects. The NK cells disclosed herein can be derived from blood (e.g., autologous or allogeneic PBMCs), NK cell lines (e.g., NK-92, NKG, YT, NK-YS, HANK-1, YTS, NKL, etc.), or differentiated stem cells (e.g., iPSCs).

[0059] I. Anti-GPRC5D antibody and anti-BCMA antibody

[0060] This document provides an anti-GPRC5D antibody or its antigen-binding fragment thereof, which contains a GPRC5D binding domain, wherein the GPRC5D binding domain includes heavy chain (VH) complementarity-determining regions (CDRs) 1, 2, and 3, and wherein the VH CDR1 has the amino acid sequence SEQ ID NO: 25, the VH CDR2 has the amino acid sequence SEQ ID NO: 26, and the VH CDR3 has the amino acid sequence SEQ ID NO: 27.

[0061] In some embodiments, the GPRC5D binding fragment of the anti-GPRC5D antibody or its antigen-binding fragment comprises a polypeptide having the amino acid sequence SEQ ID NO: 4 or 5.

[0062] In some embodiments, the GPRC5D binding fragment of the anti-GPRC5D antibody or its antigen-binding fragment comprises a polypeptide with greater than 80% sequence identity to SEQ ID NO: 4 or 5 and binding specificity to GPRC5D.

[0063] In some embodiments, the GPRC5D binding fragment of the anti-GPRC5D antibody or its antigen-binding fragment is encoded by a nucleic acid molecule having a nucleotide sequence SEQ ID NO: 31 or 32.

[0064] In some embodiments, the GPRC5D binding fragment of the anti-GPRC5D antibody or its antigen-binding fragment is encoded by a nucleic acid molecule that has greater than 80% sequence identity with the nucleotide sequence SEQ ID NO: 31 or 32 and encodes a GPRC5D binding polypeptide.

[0065] This document also provides an anti-BCMA antibody or its antigen-binding fragment comprising a BCMA-binding domain, wherein the BCMA-binding domain comprises heavy chain (VH) complementarity-determining regions (CDRs) 1, 2, and 3, and wherein the VH CDR1 has an amino acid sequence selected from SEQ ID NO: 16, 19, and 22, the VH CDR2 has an amino acid sequence selected from SEQ ID NO: 17, 20, and 23, and the VH CDR3 has an amino acid sequence selected from SEQ ID NO: 18, 21, and 24.

[0066] In some implementations, the BCMA binding domain includes VHH CDR1-3, which are SEQ ID NO: 16, 17 and 18; SEQ ID NO: 19, 20 and 21; or SEQ ID NO: 22, 23 and 24, respectively.

[0067] In some embodiments, the BCMA-binding fragment of the anti-BCMA antibody or its antigen-binding fragment comprises a polypeptide having the amino acid sequence SEQ ID NO: 1, 2, 3 or 11.

[0068] In some embodiments, the BCMA-binding fragment of the anti-BCMA antibody or its antigen-binding fragment comprises a polypeptide that has a sequence identity greater than 80% with SEQ ID NO: 1, 2, 3 or 11 and has BCMA binding specificity.

[0069] In some embodiments, the BCMA-binding fragment of the anti-BCMA antibody or its antigen-binding fragment is encoded by a nucleic acid molecule having a nucleotide sequence SEQ ID NO: 28, 29, 30 or 38.

[0070] In some embodiments, the BCMA-binding fragment of the anti-BCMA antibody or its antigen-binding fragment is encoded by a nucleic acid molecule that has greater than 80% sequence identity with the nucleotide sequence SEQ ID NO: 28, 29, 30 or 38 and encodes a BCMA-binding polypeptide.

[0071] In some embodiments, the anti-GPRC5D and / or anti-BCMA antibody or its antigen-binding fragment is a monovalent or multivalent monoclonal antibody, nanobody, scFv, Fab, F(ab)2 or Fv.

[0072] In some implementations, the anti-GPRC5D and / or anti-BCMA antibodies are humanized antibodies or chimeric antibodies.

[0073] In some embodiments, anti-GPRC5D antibodies and / or anti-BCMA antibodies or antigen-binding fragments thereof are used to construct CARs. In some embodiments, anti-GPRC5D antibodies and / or anti-BCMA antibodies or antigen-binding fragments are used to construct modified NK cells containing one or more CARs.

[0074] II. Chimeric antigen receptor (CAR) and CAR-NK cells

[0075] This article presents a bispecific CAR that can target and bind to BCMA and GPRC5D, and therefore contains at least: (a) B cell maturation antigen (BCMA) targeting domain; and (b) Targeting domain of G protein-coupled receptor C family 5 member D (GPRC5D).

[0076] The extracellular targeting domain (or so-called antigen (or ligand) binding domain) of a CAR typically links to one or more intracellular signaling components, in some cases via linkers and / or transmembrane domains. These molecules typically mimic or imitate signaling via natural antigen receptors, signaling via such receptors in combination with co-stimulatory receptors, and / or signaling via co-stimulatory receptors alone.

[0077] In some implementations, the CAR has bispecificity against specific antigens BCMA and GPRC5D, which are expressed in some or most MM cells of patients.

[0078] CARs typically contain antigen-binding molecules, such as one or more antigen-binding fragments, domains, or portions thereof, or one or more antibody variable domains, and / or antibody molecules, in their extracellular portion. In some embodiments, a CAR includes one or more antigen-binding portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from a monoclonal antibody (mAb) with a variable heavy chain (VH) and a variable light chain (VL).

[0079] Antibody fragments can be prepared using a variety of techniques, including but not limited to the proteolytic digestion of intact antibodies and the production of recombinant host cells. In some embodiments, the antibody is a recombinant-generated fragment, such as a fragment containing a non-naturally occurring arrangement, such as a fragment having two or more antibody regions or chains linked by synthetic linkers (e.g., peptide linkers), and / or may not be a fragment produced by enzymatic digestion of naturally occurring intact antibodies. In some aspects, the antibody or fragment is a VHH or scFv.

[0080] In some embodiments, the BCMA targeting domain and the GPRC5D targeting domain are independently selected from or derived from antibodies or antigen-binding fragments thereof having the desired targeting activity. In some embodiments, the targeting domain is independently selected from or derived from single-chain variable fragments (scFv), heavy-chain variable domains (VHH) of heavy-chain antibodies, and nanobodies.

[0081] In some implementations, the BCMA targeting domain and the GPRC5D targeting domain are connected in series, fused, or coupled to each other in any order, optionally with or without a connector. The arrangement of the BCMA targeting domain (a) and the GPRC5D targeting domain (b) is shown below, but is not limited to: (a)-(b), (b)-(a), (a)-L-(b), (b)-L-(a), or (a) / (b), (b) / (a), where "-" represents a bond, "L" represents a connector, and " / " represents fusion.

[0082] In some embodiments, the BCMA-targeting domain is derived from the BCMA-targeted VHH or its CDR, such as the VHH or its CDR obtained in Example 1, preferably the VHH or its CDR listed in Tables 1 and 6, more preferably the VHH or its CDR in Table 6; or a combination thereof. For example, the BCMA-targeting domain includes: VH or VHH CDR1 selected from SEQ ID NO: 16, 19 and 22; VH or VHH CDR2 selected from SEQ ID NO: 17, 20 and 23; and VH or VHH CDR3 selected from SEQ ID NO: 18, 21 and 24.

[0083] In some embodiments, the BCMA targeting domain includes VH or VHH CDR1-3, which are SEQ ID NO: 16, 17, and 18; SEQ ID NO: 19, 20, and 21; or SEQ ID NO: 22, 23, and 24, respectively. In some embodiments, the BCMA targeting domain: (a1) Contains a polypeptide having an amino acid sequence SEQ ID NO: 1, 2, 3 or 11; and / or (a2) Contains a polypeptide that has greater than 80% sequence identity with SEQ ID NO: 1, 2, 3 or 11 and has binding specificity to BCMA; and / or (a3) Encoded by a nucleic acid molecule having a nucleotide sequence SEQ ID NO: 28, 29, 30 or 38; and / or (a4) Encoded by a nucleic acid molecule that has a sequence identity greater than 80% with the nucleotide sequence SEQ ID NO: 28, 29, 30 or 38 and encodes a BCMA-binding polypeptide.

[0084] In some implementations, the GPRC5D targeting domain is derived from the GPRC5D targeting VHH or its CDR, such as the VHH or its CDR obtained in Example 2, preferably the VHH or its CDR listed in Table 6; or a combination thereof. For example, the GPRC5D targeting domain contains VH or VHH CDR1-3, which are SEQ ID NO: 25, 26 and 27, respectively.

[0085] In some implementations, the GPRC5D targets the structural domain: (b1) Contains a polypeptide having an amino acid sequence SEQ ID NO: 4 or 5; and / or (b2) Contains a polypeptide that has greater than 80% sequence identity with SEQ ID NO: 4 or 5 and has binding specificity to GPRC5D; and / or (b3) Encoded by a nucleic acid molecule having a nucleotide sequence SEQ ID NO: 31 or 32; and / or (b4) Encoded by a nucleic acid molecule that has a sequence identity greater than 80% with the nucleotide sequence SEQ ID NO: 31 or 32 and encodes a GPRC5D binding polypeptide.

[0086] In some embodiments, the CAR includes a BCMA targeting domain and a GPRC5D targeting domain connected in tandem in any order. In some embodiments, the CAR includes a polypeptide having the amino acid sequence SEQ ID NO: 12; and / or the CAR includes a polypeptide with greater than 80% sequence identity to SEQ ID NO: 12 and capable of binding BCMA and GPRC5D; and / or the CAR includes a polypeptide encoded by a nucleic acid molecule having the nucleotide sequence SEQ ID NO: 39; and / or the CAR includes a polypeptide encoded by a nucleic acid molecule with greater than 80% sequence identity to the nucleotide sequence SEQ ID NO: 3 and encoding a polypeptide capable of binding BCMA and GPRC5D.

[0087] The extracellular targeting domain (or so-called antigen (or ligand) binding domain) of a CAR typically links to one or more intracellular signaling components, in some cases via linkers and / or transmembrane domains. These molecules typically mimic or imitate signaling via natural antigen receptors, signaling via such receptors in combination with co-stimulatory receptors, and / or signaling via co-stimulatory receptors alone.

[0088] In some embodiments, the transmembrane domain is derived from a natural or synthetic source. When the source is natural, the domain is derived in some respects from any membrane-binding or transmembrane protein. The transmembrane region includes transmembrane regions derived from (i.e., including at least) the following sources: CD8, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or the α, β, or ζ chain of the T cell receptor. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain primarily comprises hydrophobic residues, such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain. In some embodiments, the CAR includes a CD8 hinge and a transmembrane region.

[0089] In some embodiments, short oligopeptide or polypeptide linkers are present, such as linkers with a length of 2-50 amino acids, such as linkers containing glycine and serine, such as glycine-serine duplexes, and a connection is formed between the transmembrane domain and the cytoplasmic signal transduction domain of the CAR.

[0090] CARs typically include at least one or more intracellular signaling components. In some embodiments, CARs include intracellular components of the TCR complex, such as the TCR CD3 that mediates T cell activation and cytotoxicity. + The CAR may contain a CD3ζ chain, such as a CD3ζ chain. Therefore, in some aspects, the antigen-binding molecule is associated with one or more cell signaling modules. In some embodiments, the cell signaling module includes a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, the CAR may also include portions of one or more other molecules, such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR includes a CD3ζ (CD3ζ) or a chimeric molecule between an Fc receptor γ and CD8, CD4, CD25, or CD16.

[0091] In some implementations, after CAR is attached, the cytoplasmic domains or intracellular signaling domains of the CAR activate at least one normal effector function or response of the NK cell.

[0092] In some embodiments, the CAR includes a signal transduction domain and / or transmembrane portion of a co-stimulatory receptor, such as 4-1BB, CD28, OX40, DAP10, and ICOS. In some aspects, the same CAR includes an activation component and a co-stimulatory component; in others, the activation domain is provided by one CAR, while the co-stimulatory component is provided by another CAR that recognizes another antigen.

[0093] In some embodiments, the intracellular signaling domain includes a CD28 transmembrane and signaling domain connected to the CD3 (e.g., CD3-ζ) intracellular domain. In some embodiments, the intracellular signaling domain includes a chimeric CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domain connected to the CD3ζ intracellular domain.

[0094] In some implementations, the CAR includes two or more co-stimulatory domains in the cytoplasmic portion that combine with an activation domain (e.g., a major activation domain). One example is a receptor that includes intracellular components of CD3-ζ and 4-1BB.

[0095] In some embodiments, the CAR comprises anti-BCMA VHH (e.g., SEQ ID NO: 1, 2, 3 or 11) and anti-HPTC5D VHH (e.g., SEQ ID NO: 4 or 5) or their tandem linkage (e.g., SEQ ID NO: 12), as well as human CD8 hinge and transmembrane region (e.g., SEQ ID NO: 7 and 8), cytoplasmic domain 4-1BB (e.g., SEQ ID NO: 9) and CD3ζ (e.g., SEQ ID NO: 10).

[0096] Using advanced genetic engineering techniques, NK cells can be engineered to express CAR, thereby forming the CAR-NK cells described in this application.

[0097] This document also provides a polynucleotide encoding the CAR of this application and a vector containing the polynucleotide for introducing it into a suitable host cell (i.e., NK cell).

[0098] III. "Armored" CAR and Armored CAR-NK Cells

[0099] Armored CARs are fourth-generation CARs that also contain intracellular domains for co-expression of regulatory small molecules, such as cytokines or chemokines. Those skilled in the art will recognize that armored CARs are a specific type of CAR, and unless otherwise stated, the term CAR encompasses various generations and types of CARs, including armored CARs.

[0100] This article also provides information on “armored” CARs and armored CAR-NK cells. As used herein, “armored CAR-NK cells” refers to NK cells that not only possess the CAR as described above, but also co-express the armor gene. Such NK cells may have one or more of the following advantages: enhanced persistence, enhanced cytotoxicity, reduced cytokine release syndrome (CRS), enhanced target cell migration, and tumor invasion.

[0101] In some implementations, the armor gene sequence is inserted into the C-terminus of the CAR and separated by a self-cleaving 2A peptide. To date, 2A-like sequences have been successfully identified in various viral mRNA molecules, including porcineteschovirus-1 2A (P2A), thosea asigna virus 2A (T2A), equinerhinitis A virus 2A (E2A), and silkworm virus (…). B. mori The softening virus (BmIFV 2A) and cytoplasmic polyhedrosis virus (BmCPV 2A).

[0102] 2A sequence-based multigene expression systems (MGES) offer several advantages: (i) they enable the expression of multiple proteins in similar amounts within the same cells and tissues due to being controlled by only one promoter; (ii) the small size of the 2A peptide allows for easy cleavage of multiple proteins while minimizing the possibility of loss of function; and (iii) proteins linked by 2A can be co-expressed across all cell types because the cleavage activity depends solely on the ribosome, which is highly structurally conserved in eukaryotes. Therefore, 2A sequence-based MGES have been widely applied in gene therapy.

[0103] The 2A peptides that can be used in this disclosure may be derived from the following group: foot-and-mouth disease virus (FMDV), encephalomyocarditis virus (EMCV), mouse encephalitis virus (TMEV), equine rhinitis virus A (ERAV), equine rhinitis virus B (ERBV), porcine cheshvirus-1 (PTV-1), and insect virus Thea asigna virus (TaV).

[0104] In some embodiments, the 2A peptide is selected from the group consisting of T2A, E2A, F2A, P2A, BmCPV2A, and BmIFV2A, such as the T2A peptide of SEQ ID NO: 15 or a sequence having at least 80% sequence identity with SEQ ID NO: 15, or a T2A peptide encoded by a nucleic acid molecule containing SEQ ID NO: 15 or a sequence having at least 80% sequence identity with SEQ ID NO: 15. Optionally, to improve the cleavage efficiency of the 2A peptide, a glycine-serine-glycine spacer (GSG) may be added to the N-terminus of the 2A peptide.

[0105] Using advanced genetic engineering techniques, NK cells can be engineered to co-express functional molecules with CARs. These functional molecules can be cytokines or chemokines, such as IL15, IL12, IL18, IL7, IL4, CXCL9, CXCL10 or any combination thereof, and can be selected according to actual needs.

[0106] Importantly, CAR-NK cells expressing multiple exogenous genes, known as "armed" CAR-NK cells or "NK cell pharmacies," promise to provide a unique and safer approach to regulating the local tumor microenvironment (TME) with fewer or no systemic adverse effects. The ability to construct "armed" CAR-NK cells offers a unique pathway for regulating the local microenvironment while avoiding systemic effects on the host.

[0107] Using advanced genetic engineering techniques, NK cells can be engineered to express CAR and armor genes, thereby forming the armored CAR-NK cells of this application.

[0108] This document also provides polynucleotides encoding the CAR and armor genes in this application, as well as vectors containing these polynucleotides for introducing them into suitable host cells (i.e., NK cells).

[0109] IV. Cell populations, cell cultures, or products

[0110] This article also provides cell populations, cell cultures, or products containing the antibodies and / or modified NK cells disclosed herein.

[0111] In some embodiments of this disclosure, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% of the cells in the cell population, cell culture, or product are the modified NK cells described in this application. In some embodiments, the cell population, cell culture, or product does not contain other cells.

[0112] In some embodiments, cell populations, cell cultures, or products can be used for disease treatment, for example, as pharmaceutical compositions and formulations. Pharmaceutical compositions and formulations typically contain one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition contains at least one other therapeutic agent.

[0113] The term "pharmaceutical preparation" refers to a preparation of such form that enables the biological activity of the active ingredient contained therein to be effective and that does not contain any other ingredients that would have unacceptable toxicity to the subject to be given the preparation.

[0114] "Pharmaceutically acceptable carriers" refer to components in a pharmaceutical preparation that are not the active ingredient and are non-toxic to the target organism. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0115] In some implementations, the choice of transport vector depends in part on the specific cells, binding molecules and / or antibodies, and / or the method of administration. Therefore, a variety of suitable formulations exist. Transport vectors are described, for example, in Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A. (1980). Pharmaceutically acceptable transport vectors are generally non-toxic to the receptor at the doses and concentrations used.

[0116] The formulation or composition may also contain more than one active ingredient capable of being used for a specific indication, disease, or condition treated with the binding molecule or cell, preferably those having complementary activity to the binding molecule or cell, wherein the respective active agents do not negatively affect each other. Such active ingredients are present in a combination of amounts that effectively achieve the intended purpose. Therefore, in some embodiments, the pharmaceutical composition also contains other pharmaceutically active substances or drugs, such as chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vincristine, etc.

[0117] In some implementations, the range of genetically engineered cells given to the subject is from about 1 million to about 100 billion cells, for example, 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined between any two of the foregoing values), for example, about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells). The number of cells, approximately 80 million cells, approximately 90 million cells, approximately 10 billion cells, approximately 25 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, or a range defined between any two of the foregoing values, and in some cases, approximately 100 million to approximately 50 billion cells (e.g., approximately 120 million cells, approximately 250 million cells, approximately 350 million cells, approximately 450 million cells, approximately 650 million cells, approximately 800 million cells, approximately 900 million cells, approximately 3 billion cells, approximately 30 billion cells, approximately 45 billion cells) or any value within these ranges, and / or that number of cells per kilogram of the subject's body weight.

[0118] The medicaments disclosed herein can be administered using standard drug delivery techniques, formulations, and / or devices. Formulations and devices for storing and administering the compositions are provided, such as syringes and vials. Cell administration can be autologous or heterologous. For example, immune-response cells or progenitor cells can be obtained from a subject and administered to the same subject or different compatible subjects. Peripheral blood-derived immune-response cells or their progeny can be administered via local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When administering therapeutic compositions (e.g., pharmaceutical compositions containing genetically modified immune-response cells), they are typically formulated as unit-dose injectable forms (solutions, suspensions, emulsions).

[0119] The formulations include those intended for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, sublingual, sublingual, or suppository administration. In some embodiments, the cell population is administered parenterally. The term "parenterally" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cell population is administered to the subject via peripheral systemic delivery through intravenous, intraperitoneal, or subcutaneous injection.

[0120] V. Treatment methods and uses

[0121] The present disclosure also provides therapeutic methods and uses for antibodies, engineered cells, cell populations, cell cultures, and products. These methods and uses may include administering antibodies, cells, or compositions containing antibodies, cells, or antibodies and cells to subjects suffering from MM disease, condition, or disorder that can be treated with antibodies or modified NK cells.

[0122] As used herein, “treatment” (and its grammatical variations, such as “treatment” or “therapeutic method”) means the complete or partial improvement or relief of a disease or condition or disorder, or symptom, side effect or outcome, or phenotype associated with it. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, relieving symptoms, reducing any direct or indirect pathological consequences of disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.

[0123] As used herein, the term "therapeutic effective amount" in the context of administration / dosage refers to the amount that effectively achieves the desired outcome (e.g., therapeutic result) within the necessary dose / quantity and time period. For a reagent (e.g., a pharmaceutical preparation), "therapeutic effective amount" refers to the amount that effectively achieves the desired therapeutic outcome (e.g., for treating a disease, condition, or disorder) and / or the therapeutic pharmacokinetic or pharmacodynamic effect within the necessary dose and time period. Therapeutic effective amount can vary depending on factors such as the subject's disease state, age, sex, weight, and the cell population administered.

[0124] This invention relates to a method for treating a subject suffering from multiple myeloma, comprising administering to the subject an effective amount of the antibody disclosed herein or modified CAR-NK cells.

[0125] Treatment includes all stages of MM (e.g., stage I, II, or III), all subtypes of MM (e.g., RRMM), and MM-related organ or tissue damage or symptoms.

[0126] Multiple myeloma staging can be determined using the International Staging System (ISS). The ISS is based on the assessment of two blood tests: β2-microglobulin (β2-M) and albumin. These two indicators have the strongest predictive power for multiple myeloma prognosis among numerous testing factors. The criteria for determining different stages of myeloma according to the International Staging System are as follows: Phase I: β2-M <3.5 mg / dL and albumin >3.5 g / dL Phase II: β2-M <3.5 mg / dL or β2-M 3.5 - 5.5 mg / dL, and albumin < 3.5 g / dL (neither Phase I nor Phase III) Phase III: β2-M >5.5 mg / dL Multiple myeloma patients are typically classified into several types. Multiple myeloma can be divided into asymptomatic and symptomatic types. Asymptomatic myeloma patients do not experience any associated organ or tissue damage or symptoms. Myeloma-related organ or tissue damage includes hypercalcemia, impaired kidney function, anemia, and bone lesions. Asymptomatic myeloma includes smoldering multiple myeloma (SMM), indolent multiple myeloma (IMM), and stage I multiple myeloma. Smoldering multiple myeloma is characterized by the presence of monoclonal proteins in the bone marrow and a slight increase in the number of plasma cells. Indolent multiple myeloma is characterized by the presence of a small amount of monoclonal proteins in the bone marrow or an increase in the number of plasma cells.

[0127] The characteristics of multiple myeloma patients also depend on their disease status. Disease status depends on whether the patient has received treatment, and if so, on the treatment outcome. Newly diagnosed patients are those with myeloma who have not yet received treatment. Patients who have received treatment are categorized as follows: Reactive disease: refers to myeloma that responds to treatment. M protein has decreased by at least 50%.

[0128] Stable / unresponsive disease: refers to myeloma that does not respond to treatment (i.e., M protein decreases less than 50%), but has not yet progressed (worsened).

[0129] Progressive disease: refers to active myeloma whose condition is worsening (i.e., elevated M protein and increased organ or tissue damage). In most cases, relapsed and / or refractory disease can be considered progressive disease.

[0130] Relapsed disease: refers to myeloma that initially responds to treatment but subsequently progresses again. Patients can be further classified as relapsed after initial treatment or relapsed after subsequent treatment.

[0131] Refractory disease: refers to myeloma that does not respond to initial treatment, and relapsed myeloma that does not respond to subsequent treatment. In the latter case, myeloma can also be referred to as relapsed refractory disease.

[0132] As used herein, “object” refers to a vertebrate, such as a mammal, such as a human or other animal, and is typically a human. In some embodiments, the object suffers from a persistent or recurrent disease, for example, after treatment with other therapies, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT. In some embodiments, the object has not yet relapsed but is determined to be at risk of relapse, such as at a high risk of relapse, and therefore is prophylactically given CAR-NK cells, for example, to reduce the likelihood of relapse or to prevent relapse.

[0133] In some embodiments, the treatment is adoptive cell therapy, in which the genetically engineered CAR-NK cells of this disclosure are given to the subject. This type of administration can promote the activation of NK cells in a targeted manner, thereby targeting and destroying cells associated with disease or ailment.

[0134] Adoptive cell therapy represents a new paradigm in cancer immunotherapy, but it may be limited by the poor persistence and function of metastatic NK cells. Natural killer (NK) cells can be xenotransplanted and have the potential to be readily available, making NK cell or CAR-NK cell adoptive cell therapy a universal option.

[0135] In some embodiments, the method includes administering antibodies, cells, or cell-containing compositions to a subject, tissue, or cells, such as a subject, tissue, or cell suffering from, at risk of suffering from, or suspected of suffering from a disease, condition, or disorder. In some embodiments, cells, populations, and compositions are administered to a subject suffering from a specific disease or condition to be treated, for example, through adoptive cell therapy, such as adoptive NK cell therapy or CAR-NK cell therapy. In some embodiments, cells or compositions are administered to a subject, such as a subject suffering from or at risk of suffering from a disease or condition. In some aspects, the method thereby treats, for example, by improving one or more symptoms of the disease or condition, such as by reducing tumor burden.

[0136] Methods for administering cells for adoptive cell therapy are known and can be used in conjunction with the methods and compositions described herein. In some embodiments, adoptive NK cell therapy is performed via autologous transfer, wherein cells are isolated from or / or otherwise prepared from a subject to receive cell therapy or from a sample of that subject. Thus, in some aspects, NK cells are derived from a subject requiring treatment (e.g., a patient) and administered to the same subject after isolation and processing.

[0137] In some embodiments, CAR-NK cell therapy is performed via allogeneic transfer, wherein cells are isolated and / or otherwise prepared from a subject other than the one about to receive or ultimately receive cell therapy (e.g., the first subject). In such embodiments, the cells are then given to a different subject of the same species, such as a second subject. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0138] Depending on the type and severity of the disease, the dosage of the cell or pharmaceutical composition may range from about 1 x 10^5 cells / kg to 3 x 10^8 cells / kg, for example, 5 x 10^5 cells / kg to 1 x 10^8 cells / kg, or 1 x 10^6 cells / kg to 3 x 10^7 cells / kg. Multiple doses may be administered intermittently, such as weekly or every three weeks. A higher initial loading dose may be given, followed by one or more lower doses.

[0139] After cells are administered to a mammal (e.g., a human), the bioactivity of the engineered cell population and / or pharmaceutical composition can be measured using any known method. Parameters evaluated include the specific binding of engineered NK cells to antigens, for example, by in vivo imaging or by ex vivo ELISA or flow cytometry. In some embodiments, the ability of engineered cells to disrupt target cells can be measured using any suitable method known in the art, such as a cytotoxicity assay. In some embodiments, cellular bioactivity is measured by determining the expression and / or secretion of certain cytokines. In some aspects, bioactivity is measured by evaluating clinical outcomes.

[0140] In some embodiments, the cell or pharmaceutical composition is administered as part of a combination therapy, for example, simultaneously with or sequentially in any order with another therapeutic intervention (e.g., another engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent).

[0141] VI. Detailed Implementation

[0142] Other embodiments of the invention are described again below. The invention also particularly provides the following items: 1. A bispecific chimeric antigen receptor (CAR) comprising (a) B cell maturation antigen (BCMA) targeting domain; and (b) Targeting domain of G protein-coupled receptor C family 5 member D (GPRC5D).

[0143] 2. The CAR as described in Project 1, wherein the BCMA targeting domain and the GPRC5D targeting domain are: (i) Independently selected from or derived from antibodies or their antigen-binding fragments; and / or (ii) Independently selected from or derived from single-chain variable fragments (scFv), heavy chain variable domains (VHH) of heavy chain antibodies, and nanobodies; and / or (iii) Connected, merged or coupled to each other in any order, optionally with or without joints.

[0144] 3. The CAR as described in Item 1, wherein the BCMA targeting domain comprises: VH or VHH CDR1 selected from SEQ ID NO:16, 19 and 22; VH or VHH CDR2 selected from SEQ ID NO:17, 20 and 23; and VH or VHH CDR3 selected from SEQ ID NO:18, 21 and 24; and / or

[0145] The BCMA targeting domain includes VH or VHH CDR1-3, which are SEQ ID NO: 16, 17 and 18; SEQ ID NO: 19, 20 and 21; or SEQ ID NO: 22, 23 and 24; and / or

[0146] The BCMA targeting domain is described below: (a1) Contains a polypeptide having an amino acid sequence SEQ ID NO: 1, 2, 3 or 11; and / or (a2) Contains a polypeptide that has greater than 80% sequence identity with SEQ ID NO: 1, 2, 3 or 11 and has binding specificity to BCMA; and / or (a3) Encoded by a nucleic acid molecule having a nucleotide sequence SEQ ID NO: 28, 29, 30 or 38; and / or (a4) Encoded by a nucleic acid molecule that has a sequence identity greater than 80% with the nucleotide sequence SEQ ID NO: 28, 29, 30 or 38 and encodes a BCMA-binding polypeptide.

[0147] 4. The CAR as described in Project 1, wherein the GPRC5D targeting domain comprises VH or VHH CDR1-3, which are SEQ ID NO: 25, 26 and 27 respectively; and / or

[0148] The GPRC5D targeting domain is described below: (b1) Contains a polypeptide having an amino acid sequence SEQ ID NO: 4 or 5; and / or (b2) Contains a polypeptide that has greater than 80% sequence identity with SEQ ID NO: 4 or 5 and has binding specificity to GPRC5D; and / or (b3) Encoded by a nucleic acid molecule having a nucleotide sequence SEQ ID NO: 31 or 32; and / or (b4) Encoded by a nucleic acid molecule that has a sequence identity greater than 80% with the nucleotide sequence SEQ ID NO: 31 or 32 and encodes a GPRC5D binding polypeptide.

[0149] 5. The CAR as described in Project 1, wherein the CAR comprises a BCMA targeting domain and a GPRC5D targeting domain connected in series in any order; and / or

[0150] The CAR contains a polypeptide having the amino acid sequence SEQ ID NO: 12; and / or

[0151] The CAR comprises a polypeptide that has greater than 80% sequence identity with SEQ ID NO: 12 and is capable of binding to BCMA and GPRC5D; and / or

[0152] The CAR comprises a polypeptide encoded by a nucleic acid molecule having the nucleotide sequence SEQ ID NO: 39; and / or

[0153] The CAR contains a polypeptide encoded by a nucleic acid molecule that has greater than 80% sequence identity with the nucleotide sequence SEQ ID NO: 39 and encodes a polypeptide capable of binding to BCMA and GPRC5D.

[0154] 6. The CAR as described in Item 1, wherein the CAR further comprises one or more domains or units selected from the group consisting of: (c) Extracellular signaling domains; (d) Extracellular hinge domain; (e) Transmembrane (TM) domain; (f) One or more intracellular signal transduction domains (ICDs); (g) Armored units; and (h) One or more independently selected joints between two domains or between a domain and a unit.

[0155] 7. The CAR as described in Project 6, wherein

[0156] (c) The extracellular signaling domain is a CD8 signal peptide; and / or

[0157] (d) The extracellular hinge domain is a CD8 hinge or an IgG4Fc hinge; and / or

[0158] (e) The transmembrane (TM) domain is selected from the group consisting of: CD8 TM, CD4 TM, CD28 TM, CD16 TM, EPOR TM, CD3ζ TM; and / or

[0159] (f) The intracellular signal transduction domain (ICD) is selected from the following group: 4-1BB (CD137), CD3ζ intracellular domain, CD8α, CD8β, co-stimulatory signal transduction domain, selected from CD27, CD28, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands, specifically binds to CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, IT GAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 ( Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162); and / or (h) The joint is independently selected from flexible joints, rigid joints, and detachable joints; and / or The connector is independently selected from serine-glycine connectors, glycine connectors, and (EAAAK). n Connectors (n is 1, 2, 3, 4, 5, 6, 7, 8, or 9), T2A connectors; and / or The connector is (G4S). n Connector, n is 1, 2, 3, 4, 5, 6, 7, 8 or 9.

[0160] 8. The CAR as described in Item 1, wherein the CAR is an armored CAR comprising one or more armored units; and / or

[0161] The armored unit includes a connector, a signal structure domain, and an armor structure domain located between the CAR and the armored unit; and / or

[0162] Wherein, the joint is a self-destructing joint; and / or

[0163] The connectors are T2A, E2A, F2A, P2A, BmCPV2A, and BmIFV2A connectors; and / or

[0164] Wherein, the armor structure domain is selected from IL15, IL12, IL18, IL7, IL4, CXCL9, CXCL10 or any combination thereof; and / or

[0165] The armor unit comprises a combination of a self-cleaving T2A connector, an IL15 CD8a signal peptide, and IL15.

[0166] 9. The CAR as described in Item 1, wherein the CAR comprises (a') and (b') or (ab'): (a') The BCMA targeting domain as defined in Project 4; and (b') The GPRC5D target domain as defined in Project 5; or (ab') The cascaded BCMA targeting domain and GPRC5D targeting domain as defined in Project 6; and The CAR mentioned above also includes: (c') The CD8 signal peptide in the extracellular signaling domain; (d')CD8TM structural domain; (e') CD8 TM in the TM structure domain; (f') the 4-1BB derived ICD and / or CD3ζ derived ICD in the ICD structural domain; (g') IL-15, the T2A autolytic linker, and the CD8a signal peptide of IL-15 in the armored unit; and (h') is a (G4S)3 connector located between (a') and (b') or in (ab').

[0167] 10. The CAR as described in Item 1, wherein the CAR comprises

[0168] (I) Possesses a combination of domains of the amino acid sequence SEQ ID NO: 7, 12, 8, 9, 10; or

[0169] (II) Having a combination of domains of the amino acid sequence SEQ ID NO: 7, 12, 8, 9, 10, 13, 14 and 15; or

[0170] (III) Combinations of domains that have a sequence identity greater than 80% with the combinations defined in (I) or (I) and are capable of combining with BCMA and GPRC5D.

[0171] 11. An anti-GPRC5D antibody or an antigen-binding fragment thereof, comprising a GPRC5D binding domain, wherein the GPRC5D binding domain comprises heavy chain (VH) complementarity-determining regions (CDRs) 1, 2, and 3, and wherein the VH CDR1 has the amino acid sequence of SEQ ID NO: 25, the VH CDR2 has the amino acid sequence of SEQ ID NO: 26, and the VH CDR3 has the amino acid sequence of SEQ ID NO: 27.

[0172] 12. Antibody or antigen-binding fragments as described in Item 11, The GPRC5D binding fragment comprises a polypeptide having the amino acid sequence SEQ ID NO: 4 or 5; and / or The GPRC5D binding fragment comprises a polypeptide with greater than 80% sequence identity to SEQ ID NO: 4 or 5 and binding specificity to GPRC5D; and / or Wherein, the GPRC5D binding fragment is encoded by a nucleic acid molecule having the nucleotide sequence SEQ ID NO: 31 or 32; and / or Wherein, the GPRC5D binding fragment is encoded by a nucleic acid molecule, wherein the nucleic acid molecule has a sequence identity greater than 80% with the nucleotide sequence of SEQ ID NO: 31 or 32 and encodes a GPRC5D binding polypeptide; and / or The antibody or antigen-binding fragment is used to construct the CAR.

[0173] 13. The antibody or antigen-binding fragment as described in Item 11, wherein the antibody or antigen-binding fragment is a monovalent or multivalent monoclonal antibody, nanobody, scFv, Fab, F(ab)2, Fv; and / or

[0174] Wherein, the antibody is a humanized antibody, a chimeric antibody; and / or

[0175] The antibody or antigen-binding fragment is used to construct modified NK cells containing one or more CARs.

[0176] 14. An anti-BCMA antibody or an antigen-binding fragment thereof comprising a BCMA-binding domain, wherein the BCMA-binding domain comprises heavy chain (VH) complementarity-determining regions (CDRs) 1, 2, and 3, and wherein the VH CDR1 has an amino acid sequence selected from SEQ ID NO: 16, 19, and 22, the VH CDR2 has an amino acid sequence selected from SEQ ID NO: 17, 20, and 23, and the VH CDR3 has an amino acid sequence selected from SEQ ID NO: 18, 21, and 24.

[0177] 15. The antibody or antigen binding fragment as described in item 14, wherein the binding domain comprises VH CDR1-3 selected from the group consisting of: SEQ ID NO: 16, 17 and 18; SEQ ID NO: 19, 20 and 21; or SEQ ID NO: 22, 23 and 24.

[0178] 16. An antibody or antigen-binding fragment as described in Item 14, wherein

[0179] Wherein, the BCMA-binding fragment comprises a polypeptide having the amino acid sequence SEQ ID NO: 1, 2, 3, or 11; and / or

[0180] Wherein, the BCMA-binding fragment comprises a polypeptide with greater than 80% sequence identity to SEQ ID NO: 1, 2, 3 or 11 and binding specificity to BCMA; and / or

[0181] Wherein, the BCMA-binding fragment is encoded by a nucleic acid molecule having the nucleotide sequence SEQ ID NO: 28, 29, 30 or 38; and / or

[0182] Wherein, the BCMA-binding fragment is encoded by a nucleic acid molecule, wherein the nucleic acid molecule has a sequence identity greater than 80% with the nucleotide sequence of SEQ ID NO: 28, 29, 30 or 38 and encodes a BCMA-binding polypeptide; and / or

[0183] The antibody or antigen-binding fragment is used to construct the CAR.

[0184] 17. The antibody or antigen-binding fragment as described in Item 14, wherein the antibody or antigen-binding fragment is a monovalent or multivalent monoclonal antibody, nanobody, scFv, Fab, F(ab)2, Fv; and / or

[0185] Wherein, the antibody is a humanized antibody, a chimeric antibody; and / or

[0186] The antibody or antigen-binding fragment is used to construct modified NK cells containing one or more CARs.

[0187] 18. One or more polynucleotide molecules encoding a CAR as described in any one of items 1-10 or an antibody or antigen-binding fragment as described in any one of items 11-17.

[0188] 19. One or more vectors comprising the following encoding polynucleotide molecules: the CAR of any one of items 1-10, the antibody or antigen-binding fragment of any one of items 11-17, or the polynucleotide molecule of item 18.

[0189] 20. A modified NK cell comprising one or more CARs as described in any one of items 1-10, one or more CARs constructed from an antibody or antigen-binding fragment as described in any one of items 11-17, one or more polynucleotide molecules encoding a CAR as described in item 18, or transduced using one or more vectors as described in item 19.

[0190] 21. Modified NK cells as described in item 20, wherein the NK cells are derived from the group consisting of: umbilical cord blood, peripheral blood, and / or placenta of vertebrates (e.g., human or rodent cells), and induced pluripotent stem cells (iPSCs); and / or

[0191] Wherein, the NK cells are autologous homologous cells or allogeneic cells; and / or

[0192] Wherein, the NK cells are transduced by the same or different vectors; and / or

[0193] The vector is selected from the group consisting of: plasmids, viruses, bacteria, bacteriophages; and / or

[0194] The modified NK cells are present in cell populations, cell cultures, or products.

[0195] 22. A product comprising one or more CARs selected from any one of items 1-10, an antibody or antigen-binding fragment selected from any one of items 11-17, one or more polynucleotide molecules selected from item 18, one or more vectors selected from item 19, and one or more modified NK cells selected from any one of items 20-21.

[0196] 23. A method for preparing modified NK cells according to any one of items 20-21, comprising the steps of: (i) modifying NK cells to contain an armored or unarmored chimeric antigen receptor (CAR) as described in any one of items 1-10 or a CAR constructed from an antibody or antigen-binding fragment as described in any one of items 11-17; and (ii) optionally, expanding and collecting the modified NK cells containing the CAR.

[0197] 24. Use of the modified NK cells described in any one of items 20-21 in the preparation of products for the treatment of multiple myeloma (MM).

[0198] 25. A method for treating multiple myeloma (MM) in a subject with this need, comprising administering an effective amount of any of the modified NK cells described in any one of items 20-21.

[0199] 26. Modified NK cells as described in any one of items 20-21 for the treatment of multiple myeloma (MM).

[0200] 27. The use as described in item 24, or the method as described in item 25, or the modified NK cells for the treatment of multiple myeloma as described in item 26, wherein said treatment is adoptive cell therapy, particularly CAR-NK adoptive cell therapy; and / or

[0201] Wherein, the MM is responsive MM, non-responsive MM, progressive MM, relapsed MM, and / or refractory MM; and / or

[0202] Wherein, the MM is an RRMM; and / or

[0203] The MM is classified as stage I, II, or III MM according to the International Myeloma Staging System.

[0204] This invention is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of embodiments of this invention. Numerical ranges include numerical values ​​that define the range. The headings provided herein are not intended to limit the various aspects or embodiments of the invention; the various aspects or embodiments of the invention can be understood in their entirety with reference to the specification.

[0205] Throughout this specification, where a composition is described as comprising an ingredient or material, it is contemplated that the composition may also consist substantially of or from any combination of the listed ingredients or materials in the embodiments, unless otherwise stated. References to “this disclosure” and “this invention”, etc., include one or more aspects taught herein, etc. The aspects taught herein are covered by the term “invention”.

[0206] Preferred embodiments described herein are selected and combined, and specific topics arising from corresponding combinations of preferred embodiments also fall within the scope of this disclosure.

[0207] The invention is further illustrated in the following examples. These examples are for illustrative purposes only and should not be construed as limiting the scope or content of the invention in any way.

[0208] All publications cited in this article and related materials therein are incorporated herein by reference in their entirety. Unless otherwise stated, all reagents are commercially available. Unless otherwise stated, all parts and percentages are by weight. Unless otherwise stated, results are shown as averages. Unless otherwise defined, abbreviations used herein are conventional.

[0209] Example

[0210] Example 1. Sequence screening of anti-BCMA VHH nanobody

[0211] A. BCMA antigen generation

[0212] The DNA sequence encoding the human BCMA extracellular domain (Uniprot number Q02223-1) was synthesized by Sangon Biotech (Shanghai, China) and then subcloned into a modified pcDNA3.3 expression vector with an MBP tag and an AVI-His tag or a human Fc tag and an AVI-His tag at the C-terminus.

[0213] Expi293 cells (Invitrogen-A14527) were transfected with the purified expression vector. After 5 days of cell culture, the supernatant was collected, and the protein was purified using a Ni-NTA column (GE Healthcare, catalog number 175248) or a Protein A column (GE Healthcare, catalog number 175438). The obtained human BCMA ECD protein was analyzed by SDS-PAGE and SEC and stored at -80°C.

[0214] B. Production of anti-BCMA benchmark (BMK) antibody (W356-BMK7-hIgG1)

[0215] The DNA sequence encoding the variable region of the baseline anti-BCMA antibody W356-BMK7 (EngMab monoclonal antibody Mab42 disclosed in patent application WO2018083204A1) was synthesized at Sangon Biotech (Shanghai, China) and then subcloned into a modified pcDNA3.3 expression vector containing the constant region of human IgG1 or IgG4.

[0216] Plasmids containing the VH and VL genes were co-transfected into Expi293 cells. Cells were cultured for 5 days, and the supernatant was collected for protein purification using a Protein A column (GE Healthcare, 175438). The resulting antibodies were analyzed by SDS-PAGE and SEC and stored at -80°C.

[0217] C. Generation of cynomolgus monkey cell lines stably expressing BCMA

[0218] The expression vector containing the cynomolgus monkey BCMA gene (XP_001106892.1) was transfected into 293F cells using Lipofectamine 2000. Cells were cultured in medium containing appropriate selection markers. After limiting dilution selection, a stable cynomolgus monkey (Cyno) BCMA high-expression cell line (W356-293F.cPro 1.P 1G5) was selected.

[0219] D. Production of anti-BCMA VHH

[0220] Anti-BCMA VHH was produced through immunization of camels and phage display technology. In short, human BCMA ECD protein (W356-hpro1.ECD.hFcAVI) tagged with hFc and mouse BCMA ECD protein (W356-mpro1.ECD.hFcAVI) were effective against alpacas (…). Vicugna pacos Subcutaneous immunization was performed. Following immunization, peripheral blood was collected, and a phage library displaying the VHH fragment was constructed. Biopanning was performed using the corresponding target ECD protein or target cell line to select positive VHH clones that bind to BCMA.

[0221] E. VHH sequencing

[0222] Positive results were selected using target-specific ELISA and FACS. E. coli Cloning, and E. coli The supernatant was sent to Biosune (Shanghai, China) for VHH gene nucleotide sequencing. The sequencing results were analyzed using CLC Main Workbench (Qiagen, Hilden, Germany).

[0223] F. Antibody humanization

[0224] To mitigate the risk of immunogenicity in clinical trials, we selected several VHHs with high affinity and specificity for BCMA for humanization. The "best-match" method was used for VHH chain humanization. The amino acid sequence of the VHH framework region was compared with the human V gene database. Then, according to the Kabat CDR definition, the human CDR sequence in the best-match result was replaced with the VHH CDR sequence, thereby generating the humanized VHH sequence. Finally, several residues in the framework region were mutated back to VHH to maintain its affinity.

[0225] The humanized VHH gene was synthesized in GENEBILZ and expressed in BL21 cells. After BCMA binding was detected using SPR, variants with suitable affinity were selected as humanized antibody lead compounds. For one VHH variant named “W3566-FP20R3-1D5”, the selected humanized antibody lead compound was W3566-FP20R3-1D5-z2 (amino acid sequence SEQ ID NO: 1; nucleotide sequence SEQ ID NO: 28).

[0226] Affinity maturation of G. VHH antibodies

[0227] To enhance the affinity of humanized VHH for human BCMA, we employed site-directed mutagenesis, mutating each amino acid in the three complementarity-determining regions (CDR1, CDR2, and CDR3) of the parental clone to 20 other amino acids. We used DNA primers containing NNS codons encoding these 20 amino acids to mutate each target CDR site. Phosphorylated degenerate primers were used in the site-directed mutagenesis reaction. 200 ng of the reaction product was electroporated into BL21 cells and expressed. We further combined point mutations in VHH that favored antigen binding to obtain a synergistic effect of enhanced affinity. These combined mutants were synthesized in GENEWIZ and expressed in BL21 cells.

[0228] The supernatant of the mutant was analyzed by SPR. After affinity maturation, the affinity-matured VHH antibody showed enhanced SPR affinity compared to the parental VHH antibody.

[0229] H. Generation of humanized VHH-Fc (human IgG1) fusion antibody

[0230] The target clone was converted into a VHH-Fc (human IgG1) fusion antibody. In this study, both the VHH antibody and the VHH-Fc fusion antibody are collectively referred to as WBP3566 antibody. In short, the VHH gene was amplified by PCR from the pET-bac vector using VHH-specific cloning primers containing the corresponding restriction endonuclease sites. This amplified gene was then cloned into a modified human hIgG1 expression pcDNA3.3 vector to construct the corresponding VHH-Fc (human IgG1) fusion antibody clone. This vector was transiently transfected into 293F or Expi293 cells for antibody expression. Cell culture supernatants containing the antibody were collected and purified using protein A affinity chromatography.

[0231] I. Complete kinetic binding affinity of VHH antibody to human and cynomolgus monkey BCMA

[0232] The binding affinity of anti-BCMA WBP3566 VHH to cynomolgus monkey BCMA and human BCMA was determined using a SPR assay on a Biacore 8K sensor. hFc-tagged cynomolgus monkey BCMA or human BCMA were captured onto a CM5 sensor chip (GE) immobilized with anti-human IgG Fc antibody. Different concentrations of WBP3566 VHH samples were injected into the sensor chip at a flow rate of 30 μL / min, with a binding phase lasting 120 s, followed by dissociation for 300–3000 s. After each binding cycle, the chip was regenerated with 10 mM glycine solution (pH 1.5).

[0233] Sensor images were obtained by subtracting the blank surface and buffer channels from the test sensor images. Langmür analysis was used, and the experimental data were fitted using a 1:1 model. The molar concentration of the WBP3566 VHH sample was calculated using a molecular weight of 15 kDa. Table 1 shows the binding affinity of the WBP3566 VHH antibody W3566-FP20R3-1D5-z2 and its affinity-matured VHH variants to human and monkey BCMA, including the affinity-matured variants W3566-FP20R3-1D5-z2-m17 (amino acid sequence SEQ ID NO: 2; nucleotide sequence SEQ ID NO: 29) and W3566-FP20R3-1D5-z2-m52 (amino acid sequence SEQ ID NO: 3; nucleotide sequence SEQ ID NO: 30).

[0234] Table 1. Complete kinetic binding affinity of anti-BCMA VHH to cynomolgus monkey and human BCMA

[0235] J. W3566 VHH-Fc (human IgG1) fusion antibody binds to cell surface BCMA

[0236] The test antibody and the isotype control antibody were serially diluted and incubated with NCI-H929 cells expressing human BCMA or cynomolgus monkey BCMA-transfected cells, respectively. The binding of the antibody to BCMA on the cell surface was then detected using the secondary antibody goat anti-human IgG Alexa Fluor647 (Jackson, catalog number: 109-605-098).

[0237] Figure 1 A and 1B show the binding of W3566-FP20R3-1D5-z2 and its affinity-matured VHH variant to BCMA on the cell surface. Binding of W3566 antibody to NCI-H929 cells (EC) 50 The values ​​range from 2 to 6 nM, while the EC values ​​of cynomolgus monkey BCMA are similar. 50 The value ranges from 0.7 to 1.4 nM.

[0238] The experimental data above confirm that, through the above method, we have successfully obtained anti-BCMA VHH nanobodies with high affinity for BCMA on the cell surface.

[0239] Example 2. Sequence screening of anti-GPRC5D VHH nanobody

[0240] A. Generation of anti-GPRC5D baseline antibody

[0241] Anti-GPRC5D reference antibodies W3XX109-cAb1 and W3XX109-cAb2 were prepared based on the sequence GCDB72 disclosed in Janssen patent US20180037651A1 and the sequence 5F11-TCB disclosed in Roche patent US20220259318, respectively. DNA sequences encoding the variable regions of GCDB72 and 5F11-TCB were subcloned into a modified pcDNA3.4 expression vector containing the constant region of human IgG1. Human IgG1 isotype control antibodies targeting the envelope glycoprotein GP120 were provided by WuXi Biologics.

[0242] B. Cell lines / banks and tumor cells

[0243] Cell lines expressing human GPRC5D (WBP3XX109-CHOK1.hPro1.C7 and WBP3XX109-293F.hPro1.B8) were generated. In short, following the manufacturer's protocol, the pcDNA3.3 expression vector containing full-length human GPRC5D (NM_018654.1) was transfected into CHO-K1 or 293F cells using the Lipofectamine 2000 transfection kit. 48–72 hours after transfection, the transfected cells were cultured in medium containing blastcinon for selection, and then human GPRC5D expression was tested by flow cytometry. Cell lines expressing human GPRC5D were obtained using limiting dilution or the BD FACSMelody™ cell sorter.

[0244] A cynomolgus monkey (Cyno) GPRC5D expression cell line (WBP3XX109-293F.cPro 1.E 1) was generated. In short, following the manufacturer's protocol, the pcDNA3.3 expression vector containing full-length cynomolgus monkey GPRC5D (XM_005570192.2) was transfected into 293F cells using the Lipofectamine 2000 transfection kit. 48–72 hours after transfection, the transfected cells were cultured in medium containing blastomycin for selection, and GPRC5D expression was detected by fluorescence-activated cell sorting (FACS). The cynomolgus monkey GPRC5D expression cell line was obtained using the BD FACSMelody™ cell sorter.

[0245] Cells expressing human GPRC5A were generated. In short, following the manufacturer's protocol, 293F cells were transfected with the pcDNA3.3 expression vector containing full-length human GPRC5A (NM_003979.3) and a Flag tag using the Lipofectamine 2000 (Thermo Fisher Scientific) transfection kit. Transfected cells were collected 48–72 hours after transfection, and Flag tag expression was detected by FACS.

[0246] Human multiple myeloma cells MM.1S (ATCC, CRL-2974), MM.1R (ATCC, CRL-2975), human acute lymphoblastic leukemia cells Nalm-6 (ATCC, CRL-3273), and Chinese hamster ovary cells CHO-K1 (ATCC, CCL-61) were purchased from ATCC. Human multiple myeloma cells OPM-2 (DSMZ, ACC 50) were purchased from DSMZ. Human renal epithelial cells 293F (Invitrogen, R79007) and Expi293 (Invitrogen, A14635) were purchased from Invitrogen.

[0247] C. Immunological and serum antibody titer detection

[0248] To induce a humoral immune response against human GPRC5D in camels, alpacas were immunized with 1 mg of a plasmid expressing full-length human GPRC5D and an adjuvant (CpG DNA) via intradermal and intramuscular injections (ID and IM) every two weeks for a total of six injections. When serum titers of anti-human GPRC5D reached adequate levels, alpacas were given two subcutaneous booster immunizations, each using 293F cells expressing human GPRC5D and supplemented with an IFA adjuvant.

[0249] Anti-human and cynomolgus monkey GPRC5D specific antibody titers were determined using WBP3XX109-CHOK1.hPro1.C7 and WBP3XX109-293F.cPro1.E1 cells. Briefly, WBP3XX109-CHOK1.hPro1.C7 and WBP3XX109-293F.cPro1.E1 cells (1×10⁻⁶) were used. 5 Cells (100 cells / well) were incubated with different dilutions of immune serum at 4°C for 1 hour. After washing the cells twice with 1×PBS / 2% BSA, goat anti-alpaca IgG (H+L)-RPE (Antibodies-online, ABIN3045709) was added. The cells were incubated at 4°C in the dark for 0.5 hours. After washing the cells twice with 1×PBS / 2% BSA, the cells were analyzed by flow cytometry (BD Canto II).

[0250] D. Phage library construction

[0251] Blood samples were collected from alpacas after their final booster immunization. Peripheral blood mononuclear cells (PBMCs) were purified by density gradient centrifugation using Ficoll-Paque (GE Healthcare, catalog number GE-17-1440-03). Total mRNA was extracted from these PBMCs and reverse transcribed into cDNA using oligo-dT primers and the SuperMix system (Ingenium, catalog number 18080400) with SuperScript III first-strand synthesis, as recommended by the manufacturer.

[0252] Using purified cDNA as a template, a library of Ig heavy chain-encoding gene segments was amplified using signal peptide domain-specific primers and CH2 domain-specific primers. This amplification reaction yielded PCR fragments of approximately 900 bp (corresponding to conventional IgG) and 700 bp (corresponding to heavy chain IgG lacking the CH1 domain). These two types of heavy chain-encoding genes were then separated by size using agarose gel electrophoresis, and the gene encoding only heavy chain IgG was purified using a QIAquick gel extraction kit (Qiagen, catalog number 28706). Using the purified fragment as a template, a VHH library was amplified using frame 1 (FR1) and frame 4 (FR4) specific primer pairs. This amplification process introduced an Sfi I restriction site at the 5' end of FR1 and a Not I restriction site at the 3' end of FR4. The approximately 300–400 bp VHH gene library obtained from PCR amplification was loaded onto an agarose gel and purified using a QIAquick gel extraction kit (Qiagen, catalog number 28706). The purified fragment was double-digested with Sfi I and Not I, and then purified using the QIAquick PCR purification kit (Kiagen, catalog number 28106). Finally, the VHH gene fragment was ligated into the phage vector pFL249 and introduced into *E. coli* TG1 cells via electroporation. After transformation, TG1 cells were cultured in SOC medium at 37°C and 200 rpm for 1 hour with shaking, then plated onto solid 2YT medium supplemented with 100 μg / mL carbenicillin and 1% (w / v) glucose, and incubated overnight at 37°C. The next day, colonies were scraped into liquid 2YT medium supplemented with 1 / 3 (v / v) 80% glycerol and stored at -80°C.

[0253] E. Screening for phage display of anti-GPRC5D specific VHH fragment

[0254] Frozen library cells were thawed and grown in 2YT medium supplemented with 100 μg / mL carbenicillin and 1% (w / v) glucose. Cell cultures were incubated in a shaker at 37°C and 220 rpm until the cell density reached OD600 = 0.4–0.6. Cells were infected with M13KO7 helper phage and incubated at 37°C for 1 hour, followed by IPTG supplementation and overnight incubation. The supernatant was collected by centrifugation and precipitated with PEG / NaCl. The precipitated phage was resuspended in PBS and its titer was determined.

[0255] To screen VHH fragments that can effectively bind to GPRC5D, a cell-based screening method was used. The screening strategy is shown in Table 2.

[0256] For cell-based panning, GPRC5D engineered cells and tumor cells were incubated with phage libraries at 4°C in the dark for 2 hours. After thorough washing with 1×PBS / 5% FBS, non-specifically adsorbed phages were discarded, and target-specifically bound phages were eluted with 0.1 M glycine-HCl (pH 2.2), followed by neutralization with 1 M Tris-HCl (pH 8.0) for use in infecting TG1 cells in the exponential growth phase. Infected TG1 cells were rescued with M13KO7 helper phage for the next round of selection and / or plated on agar plates for selection of single clones.

[0257] Table 2. Selection Strategy

[0258] F. VHH fragment expression, screening, and VHH-Fc protein production

[0259] After the required selection, the VHH fragment was subcloned into an expression vector containing a hexahistine tag and a c-Myc tag gene. The vector containing the subclone was transformed into *E. coli* BL21(DE3) competent cells. Single colonies were picked and expressed in ZYM-5052 medium. The bacterial culture supernatant was collected for selection.

[0260] The binding of human GPRC5D in the expression supernatant was screened using MM.1S cells by flow cytometry. In short, 1×10⁻⁶ cells were used... 5 MM.1S cells were incubated with VHH expression supernatant at 4°C for 1 hour in the dark, followed by washing three times (1×PBS / 1% BSA). The cells were then incubated with a 1:800 dilution of goat anti-cMyc-RPE antibody (Bethyl) at 4°C for 30 minutes in the dark. The sample was washed and resuspended in 1×PBS / 1% BSA. The cell suspension was then analyzed on a BD FACS array.

[0261] All positive clones were sent for sequencing. The antibodies were converted into VHH-Fc (human IgG1) fusion antibodies. In short, the DNA sequence encoding VHH was synthesized by GENEWIZ (Suzhou, China) and then cloned into a modified pcDNA3.4 expression vector containing a human IgG1 Fc fragment to construct the corresponding VHH-Fc chimeric antibody clone. This vector was transiently transfected into Expi293 cells (Ingenie, catalog number A14635) for antibody expression. The cell culture supernatant containing the antibody was collected and purified using protein A affinity chromatography. Several VHH-Fc fusion antibodies were then further tested. One selected VHH-Fc fusion antibody was named W306109-P7R2-1G4-uIgG1, and the corresponding VHH binding is W306109-P7R2-1G4 (amino acid sequence SEQ ID NO: 4; nucleotide sequence SEQ ID NO: 31).

[0262] G. Human GPRC5D cell binding assay

[0263] WBP3XX109-CHOK1.hPro1.C7 cells and multiple myeloma cells were incubated with different concentrations of VHH-Fc antibody at 4°C for 1 hour. After washing the cells with 1×PBS / 1% BSA, PE-labeled goat anti-human antibody (Jackson ImmunoResearch, catalog number 109-115-098) was added. The cells were incubated at 4°C in the dark for 1 hour. Anti-human GPRC5D reference BMK antibodies W3XX109-cAb1 and W3XX109-cAb2 were used as positive controls. Human IgG1 isotype antibody was used as a negative control. The cells were then washed and resuspended in 1×PBS / 1% BSA. The mean fluorescence intensity (MFI) of the cells was measured using a flow cytometer (BD Canto II) and analyzed using FlowJo software.

[0264] The results of antibody binding to MM.1R or OPM-2 cells are as follows: Figure 2 Figures A and 2B show results for W306109-P7R2-1G4-uIgG1 and BMK only (in these figures and subsequent graphs). The results indicate that W306109-P7R2-1G4-uIgG1 specifically binds to GPRC5D-positive MM.1R and OPM-2 cells, but not to GPRC5D-negative Nalm-6 cells. Figure 2 C). The antibody binding results are summarized in Table 3.

[0265] Table 3. EC2 cell binding assays of MM.1R and OPM-2 50Summary of highest MFI values

[0266] Note: NA = unavailable due to the lack of a complete curve; EC 50 Half-maximum effective concentration; MFI: mean fluorescence intensity

[0267] H. Cross-species combination experiments

[0268] Cells transduced with cynomolgus monkey GPRC5D were incubated with different concentrations of VHH-Fc antibody at 4°C for 1 hour. After washing the cells with 1×PBS / 1% BSA, PE-labeled goat anti-human antibody (Jackson Immunolab Laboratories, catalog number 109-115-098) was added. The cells were incubated at 4°C in the dark for 1 hour. The cells were then washed and resuspended in 1×PBS / 1% BSA. The mean fluorescence intensity (MFI) of the cells was measured using a flow cytometer (BD Canto II), and analyzed using FlowJo software. The binding results of the antibody to cynomolgus monkey GPRC5D are shown below. Figure 3 As shown, a summary of the results is presented in Table 4.

[0269] Table 4. EC5D binding assay in cynomolgus monkeys 50 Summary of highest MFI values

[0270] Note: NA = unavailable due to the lack of a complete curve; EC 50 Half-maximum effective concentration; MFI: mean fluorescence intensity

[0271] I. Paralog binding assay

[0272] Human GPRC5A-positive 293F cells were incubated with different concentrations of VHH-Fc antibody at 4°C for 1 hour. After washing the cells with 1×PBS / 1% BSA, PE-labeled goat anti-human antibody (Jackson Immunolab Laboratories, catalog number 109-115-098) was added. The cells were incubated at 4°C in the dark for 1 hour. The cells were then washed and resuspended in 1×PBS / 1% BSA. The mean fluorescence intensity (MFI) of the cells was measured using a flow cytometer (BD Canto II), and analyzed using FlowJo software.

[0273] W306109-P7R2-1G4-uIgG1 showed almost no binding to human GPRC5A, while W3XX109-cAb2 showed a small amount of nonspecific binding to GPRC5A. The binding results are shown in Table 5.

[0274] Table 5. Summary of mean fluorescence intensity (MFI) in human GPRC5A binding assay

[0275] J. Antibody Humanization

[0276] To mitigate the risk of immunogenicity in clinical trials, we selected several VHH conjugates with high affinity and specificity for GPRC5D for humanization. Subsequently, we tested the humanized VHH candidates on human GPRC5D-expressing cell lines. For the VHH conjugate “W306109-P7R2-1G4”, a final humanized variant with appropriate affinity, designated “W306109-P7R2-1G4-z02” (amino acid sequence SEQ ID NO: 5; nucleotide sequence SEQ ID NO: 32), was selected as a humanized antibody lead.

[0277] The experimental data above confirm that, through the above method, we have successfully obtained anti-GPRC5D VHH nanobodies with high specificity and affinity for GPRC5D on the cell surface.

[0278] Table 6 lists some exemplary VHH mAb CDR sequences generated for human BCMA or human GPRC5D using the methods of Examples 1 and 2.

[0279] Table 6. CDR sequences for VHH mAb generated by human BCMA or human GPRC5D

[0280] Note: CDR (Complementary Decision Region) is defined according to the Kabat+IMGT numbering scheme.

[0281] Example 3. Generation of anti-BCMA×GPRC5D chimeric antigen receptor (CAR) and cells expressing anti-BCMA×GPRC5D CAR

[0282] Chimeric antigen receptors (CARs) were engineered to incorporate a VHH targeting BCMA (from Example 2) and / or a VHH targeting GPRC5D (from Example 3). A multinucleotide construct encoding a dual-tandem CAR was generated, which encodes an antigen-binding domain comprising an anti-BCMA VHH and an anti-GPRC5D VHH, wherein the two VHHs are linked via a (G4S)3 linker (amino acid sequence SEQ ID NO: 6; nucleotide sequence SEQ ID NO: 33). Figure 4 For single BCMA CAR or single GPRC5D CAR, the antigen-binding domain contains only anti-BCMA VHH or only anti-GPRC5D VHH.

[0283] Each generated CAR construct contains a human CD8 signal peptide (amino acid sequence SEQ ID NO: 7; nucleotide sequence SEQ ID NO: 34); a single VHH or tandem VHH antigen-binding domain; a human CD8 hinge and transmembrane (TM) domain (amino acid sequence SEQ ID NO: 8; nucleotide sequence SEQ ID NO: 35); a human 4-1BB-derived intracellular (ICD) signaling domain (amino acid sequence SEQ ID NO: 9; nucleotide sequence SEQ ID NO: 36); and a human CD3ζ-derived intracellular signaling domain (amino acid sequence SEQ ID NO: 10; nucleotide sequence SEQ ID NO: 37). Figure 4 ).

[0284] To screen for the optimal tandem BCMAxGPRC5D dual CAR, various BCMA-targeted VHHs from Example 2 and GPRC5D-targeted VHHs from Example 3 were selected and combined. Approximately 20 BCMA×GPRC5D combinations were tested, including two sequences: BCMA-VHH~Connector~GPRC5D-VHH-Hinge-TM-ICD and GPRC5D-VHH~Connector~BCMA-VHH-Hinge-TM-ICD. After functional analysis and screening, the optimal tandem BCMA×GPRC5D CAR was verified, namely BCMA-1D5(W3566-FP20R3-1D5-z2-m17)~(G4S)3Connector~GPRC5D-1G4(W306109-P7R2-1G4-z02)-Hinge-TM-ICD. Table 7 lists the sequences of the single BCMA-1D5 (amino acid sequence SEQ ID NO: 11; nucleotide sequence SEQ ID NO: 38) CAR, the single GPRC5D-1G4 (amino acid sequence SEQ ID NO: 5; nucleotide sequence SEQ ID NO: 32) CAR, and the double tandem BCMA-1D5xGPRC5D-1G4 (amino acid sequence SEQ ID NO: 12; nucleotide sequence SEQ ID NO: 39) CAR.

[0285] The BCMA-BMK CAR sequence is derived from LCAR-B38M BCAR003 (as disclosed in patent WO2017025038), which contains a human CD8 signal peptide, two bilateral VHH binding domains, a human CD8 hinge and a transmembrane domain; an intracellular signal transduction domain derived from human 4-1BB and an intracellular signal transduction domain derived from human CD3ζ.

[0286] The GPRC5D-BMK sequence is derived from MCARH109 (as disclosed in patent WO2020092854, abbreviated as CAR203), which contains a human CD8 signal peptide; an antigen-binding domain containing scFv, wherein the variable heavy chain (VH) and the variable light chain (VL) are linked by a linker; a long immunoglobulin-derived spacer domain (hinge-CH2-CH3) containing CH2 modification to restrict Fc receptor binding; a human CD28-derived transmembrane domain; a human 4-1BB-derived intracellular signal transduction domain; and a human CD3ζ-derived intracellular signal transduction domain.

[0287] Table 7. Components and SEQ ID NO of the generated CAR construct

[0288] For fourth-generation CAR-NK cells carrying the IL15 armor gene, the nucleic acid construct encoding the CAR is followed by sequences encoding the human CD8 signal peptide (amino acid sequence SEQ ID NO: 13; nucleotide sequence SEQ ID NO: 40) and codon-optimized human IL15 (amino acid sequence SEQ ID NO: 14; nucleotide sequence SEQ ID NO: 41). The IL15 armor gene and the CAR sequence are separated by a self-cleaving T2A sequence (amino acid sequence SEQ ID NO: 15; nucleotide sequence SEQ ID NO: 42). Figure 4 (A and 4B).

[0289] The nucleic acid construct was cloned into the pMSCV retroviral expression vector (Miaolingbio, P10650) for cell transduction. Retroviruses were produced using 293T cells, and their titers were determined.

[0290] For the transduction of primary human NK cells, primary human NK cells were isolated from PBMCs (peripheral blood mononuclear cells) obtained from healthy donors. NK cells were stimulated with feeder cells (K562-mbIL21-41BBL) in the presence of recombinant IL-2. After expansion for 5-6 days, NK cells were transduced with a retrovirus pre-coated with RetroNectin. Subsequently, the transduced NK cells were expanded in the presence of recombinant IL-2 and used for functional assays or cryopreservation approximately 7-10 days post-transduction.

[0291] For the transduction of primary human T cells, primary human T cells were also isolated from PBMCs obtained from healthy donors. T cells were stimulated with T CellTransAct (Miltenyi Biotec). After expansion for 3 days in the presence of recombinant IL-2, the T cells were transduced using a retrovirus with the aid of the transduction enhancer Vectofusin-1 (Miltenyi Biotec). The transduced T cells were then expanded in the presence of recombinant IL-2 and used for functional assays or cryopreservation approximately 7–12 days post-transduction.

[0292] The expression rates of BCMA-1D5xGPRC5D-1G4 tandem CAR, BCMA-1D5 monoCAR, BCMA-BMK CAR, and GPRC5D-1G4 monoCAR were detected using MonoRab™ rabbit anti-cameloid VHH mixed antibody [PE] (Genscript, A02018) or MonoRab™ rabbit anti-cameloid VHH mixed antibody [iFluor 647] (Genscript, A02019). The expression rate of GPRC5D-BMK (MCARH109, ​​IgG4 hinge) was detected using primary antibody anti-IgG4 antibody [EP4420] (Abcam, ab109493) conjugated with secondary antibody Alexa Fluor 647 to goat anti-rabbit IgG (H+L) (Jackson Immunolabs, 111-605-144).

[0293] The results showed that after retroviral transduction, all single and tandem CARs were highly expressed on NK cells. Figure 5 ).

[0294] Example 4. In vitro activity of BCMAxGPRC5D-targeted CAR-NK cell therapy

[0295] For CAR-NK cells incorporating BCMAxGPRC5D tandem CAR, BCMA mono-CAR, BCMA BMK, GPRC5D mono-CAR, and GPRC5D BMK, their cytotoxic functional activity and ability to induce cytokine production in the presence of antigen were tested. To generate CAR-NK cells, NK cells were isolated, stimulated, transduced, and expanded as described in Example 3.

[0296] A. Cytotoxicity

[0297] The expression of GPRC5D and BCMA in a group of multiple myeloma cell lines was assessed by flow cytometry. Figure 6 A). The results showed that the MM cell line expressed GPRC5D and BCMA.

[0298] To understand the specificity of anti-BCMA and anti-GPRC5D CARs, GPRC5D or BCMA was knocked out, respectively, in OPM2-luciferase-GFP cells using the CRISPR-Cas9 gene editing tool. Figure 6 As shown in B and 6C, flow cytometry analysis confirmed that OPM2 GPRC5D KO cells lacked GPRC5D expression (without affecting BCMA expression), and OPM2 BCMA KO cells lacked BCMA expression (without affecting GPRC5D expression). OPM2 wild-type cells served as a control, showing GPRC5D and BCMA expression.

[0299] CAR-NK cells were co-cultured with engineered human tumor cell lines OPM2, NCI-H929, and RPMI8226 (all expressing endogenous BCMA and GPRC5D) expressing green fluorescence (ZsGreen or GFP) at different effector cell to target cell (E:T) ratios for 24 or 48 hours. Target cell killing was determined using the Operetta CLS high-content analysis system (PerkinElmer) to detect green fluorescence, and the killing was normalized relative to the killing of target tumor cells cultured alone (N=3, mean ± SEM). Figure 7 As shown in A, 7B, and 7C, NK cells expressing the anti-BCMAxGPRC5D tandem lead CAR induced cytotoxicity across a wide range of effector-to-target cell ratios. These cytotoxicity results were comparable to those of BCMA-1D5 monoCAR, BCMA-BMK, GPRC5D-1G4 monoCAR, and GPRC5D-BMK CAR-NK cells.

[0300] On the other hand, when co-cultured with BCMA-KO OPM2 or GPRC5D-KO OPM2 cells, tandem BCMA×GPRC5D dual CAR-NK cells showed better target coverage than BCMA or GPRC5D single CAR-NK cells. Figure 7 As shown in D and 7E, BCMA-1D5 and BCMA-BMK single CAR-NK cells did not exhibit any specific cytolytic activity against BCMA-KO OPM2 cells, while GPRC5D-1G4 and GPRC5D-BMK single CAR-NK cells also did not respond to GPRC5D-KO OPM2 cells. In contrast, tandem BCMA×GPRC5D dual CAR-NK cells showed strong cytolytic activity against both KO cell lines.

[0301] In addition, in the cytotoxicity assay, we used the OPM2 hybrid model, which contained 50% BCMA-KO OPM2-ZsGreen cells and 50% GPRC5D-KO OPM2-ZsGreen cells. Tumor lysis of CAR-NK cells in the OPM2 hybrid model was detected using the Incucyte S3 live cell analysis system (Sartorius). Figure 8 (where the Y-axis represents the GFP signal intensity of the remaining surviving target cells). Figure 8 As shown, only BCMA×GPRC5D tandem lead CAR-NK can clear tumor cells in the OPM2 hybrid model, while BCMA or GPRC5D single CAR and BMK CAR-NK failed to completely clear the tumor.

[0302] In summary, tandem BCMAxGPRC5D CAR-NK has better target coverage than single-target CAR-NK, providing a method to combat MM tumor heterogeneity.

[0303] B. Cytokine secretion

[0304] BCMAxGPRC5D CAR-NK cells were co-cultured with OPM2 or NCI-H929 MM cells at a ratio of 1:1 or 0.5:1 for 1 or 2 days, and the supernatant was collected for IFN-γ detection.

[0305] like Figure 9 As shown in A and 9B, after co-culturing with OPM2 or NCI-H929 MM cell lines, the IFN-γ secretion of tandem leader BCMAxGPRC5DCAR-NK, BCMA single CAR-NK, and GPRC5D single CAR-NK was comparable.

[0306] Example 5. IL15 armor gene enhances the cytotoxicity and persistence of CAR-NK cells.

[0307] CAR-NK and CAR-T cells were generated as described in Example 3. The cytotoxicity of CAR-NK and CAR-T cells was assessed 1 or 2 days after cell thawing. Figure 10 As shown in Figure A, tandem-guided BCMAxGPRC5D CAR-NK cells exhibited stronger cytotoxicity than control NK cells, and the IL15 armor gene further significantly enhanced the cytotoxicity of tandem-guided CAR-NK cells. The tumor lysis activity of IL15-armored tandem-guided BCMAxGPRC5D CAR-NK cells was also significantly stronger than that of tandem-guided BCMAxGPRC5D CAR-T or BCMA-BMK CAR-T. Figure 10 B).

[0308] Compared to CAR-T cells, the insufficient persistence of second-generation CAR-NK cells (lacking the IL-15 armor gene) is a drawback. The introduction of the IL-15 armor gene significantly improved the persistence of fourth-generation CAR-NK cell products. We conducted a continuous killing assay lasting up to 300 hours using the Incucyte S3 live cell analysis system. Figure 11 Following continuous addition of target cells OPM2-ZsGreen and several rounds of stimulation, the IL-15-armored tandem leader BCMAxGPRC5D CAR-NK demonstrated superior durable cytotoxicity compared to unarmored IL-15 CAR-NK, BCMAxGPRC5D CAR-T, or BCMA-BMK CAR-T. This enhanced durability brought about by the IL-15 armor gene holds promise for further enhancing the clinical benefits of CAR-NK.

[0309] Despite the high cytotoxicity and persistence of the IL15 armor gene, it did not significantly affect IFN-γ release from CAR-NK cells, and compared with CAR-T cells, IL15-armored CAR-NK cells still secreted significantly less IFN-γ upon target cell stimulation. Figure 12 This is consistent with the fact that CAR-NK cells cause far less cytokine release syndrome (CRS) than CAR-T cells in clinical practice, which is a significant advantage of CAR-NK cells in terms of clinical safety.

[0310] Example 6. In vivo activity of BCMAxGPRC5D CAR-NK

[0311] The OPM2 human myeloma cell line xenograft model induces bone marrow-dominant disease and was used to evaluate the in vivo efficacy of cryopreserved BCMAxGPRC5D CAR-NK cell therapy.

[0312] To NPG (NOD-Prkdc) scid IL2rg null Immunodeficient mice (female, 8-10 weeks old, weighing 22-25 grams, 4 mice per group, purchased from Beijing Vitalstar Biotech Co., Ltd.) were injected via tail vein with 1×10 6 One OPM2-Luc (firefly luciferase) cell was implanted and allowed to expand for approximately 8 days, then 1 × 10⁶ cryopreserved cells were injected via tail vein. 7A single treatment was administered using IL15-armored BCMAxGPRC5D CAR-NK, IL15-free BCMAxGPRC5D CAR-NK, NK-mimicking cells (CD19 CAR-NK with the IL15 armor gene), or a carrier (200 μl buffer). CAR-NK cells were directly injected after thawing to simulate future clinical use. Tumor burden was monitored using luciferase activity bioluminescence imaging (BLI). Figure 13 A and 13B).

[0313] As shown in the figure, compared with the simulated NK cell, BCMAxGPRC5D CAR-NK (without IL15) exhibited moderate but significant tumor suppressor activity; while compared with the simulated NK cell and the IL15-armored BCMAxGPRC5D CAR-NK, the IL15-armored BCMAxGPRC5D CAR-NK showed significantly enhanced tumor suppressor activity in the OPM2-luc mouse model. Mouse model data demonstrate the targeting activity of BCMA×GPRC5D CAR and the further enhancement of CAR-NK activity in vivo by the IL15 armor gene.

[0314] This invention is not limited to the embodiments described herein, which are merely intended to illustrate individual aspects of the invention, and any functionally equivalent forms are also within the scope of this invention. Based on the foregoing and teachings, various modifications to the compositions and methods of this invention, in addition to those described herein, will be apparent to those skilled in the art and are also intended to fall within the scope of this invention. Such modifications or other embodiments may be implemented without departing from the true scope and spirit of this invention.

[0315] Appendix: Specific sequences in this application

[0316] SEQ ID NO 1; PRT; W3566-FP20R3-1D5-z2 (Anti-BCMA VHH)

[0317] EVQLVESGGGLVQPGGSLRLSCAASGSIDSINAIAWYRQAPGKQRELVSFISSGGFPDYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCNAERVWGGKIYNYWGQGTMVTVSS

[0318] SEQ ID NO 2; PRT; W3566-FP20R3-1D5-z2-m17 (Anti-BCMA VHH)

[0319] EVQLVESGGGLVQPGGSLRLSCAASGSIDSINAIAWYRQAPGKQRELVSFISSGGFPDYADWVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCNAEEVWGGKIYNYWGQGTMVTVSS

[0320] SEQ ID NO 3; PRT; W3566-FP20R3-1D5-z2-m52 (anti-BCMA VHH)

[0321] EVQLVESGGGLVQPGGSLRLSCAASGSIWSINAIAWYRQAPGKQRELVSFISSGGFPDYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCNAERVWGGKIYNYWGQGTMVTVSS

[0322] SEQ ID NO 4; PRT; W306109-P7R2-1G4 (anti-GPRC5D VHH)

[0323] QVQLVESGGGLVQAGGSLRLSCAISGRTRSGADMAWYRQAPGKEREFVAAITWNDGLTYYADSVKGRFAISRDDAKNTLLTLQMNSLKPEDTAIYYCNNRFRARWWDWGQGTQVTVSS

[0324] SEQ ID NO 5; PRT; W306109-P7R2-1G4-z02 (anti-GPRC5D VHH)

[0325] EVQLVESGGGLVKPGGSLRLSCAASGRTRSGADMAWYRQAPGKEREFVAAITWNDGLTYYADSVKGRFTISRDDSKNTLYLQMNSLKPEDTAVYYCNNRFRARWWDWGQGTMVTVSS

[0326] SEQ ID NO 6; PRT; (G4S)3 linker

[0327] GGGGSGGGGSGGGGS

[0328] SEQ ID NO 7; PRT; human CD8 signal peptide

[0329] MALPVTALLLPLALLLHAARP

[0330] SEQ ID NO 8; PRT; Human CD8 hinge and transmembrane (TM) domain

[0331] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

[0332] SEQ ID NO 9; PRT; Human 4-1BB-derived intracellular (ICD) signal transduction domain

[0333] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0334] SEQ ID NO 10; PRT; Human CD3ζ-derived intracellular signal transduction domain

[0335] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0336] SEQ ID NO 11; PRT; BCMA-1D5, W3566-FP20R3-1D5-z2-m17

[0337] EVQLVESGGGLVQPGGSLRLSCAASGSIDSINAIAWYRQAPGKQRELVSFISSGGFPDYADWVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCNAEEVWGGKIYNYWGQGTMVTVSS

[0338] SEQ ID NO 12; PRT; BCMA-1D5~ (G4S)x3 connector~ GPRC5D-1G4

[0339] EVQLVESGGGLVQPGGSLRLSCAASGSIDSINAIAWYRQAPGKQRELVSFISSGGFPDYADWVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCNAEEVWGGKIYNYWGQGTMVTVSSGGGGSG GGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGRTRSGADMAWYRQAPGKEREFVAAITWNDGLTYYADSVKGRFTISRDDSKNTLYLQMNSLKPEDTAVYYCNNRFRARWWDWGQGTMVTVSS

[0340] SEQ ID NO 13; PRT; Human CD8a signal peptide of IL15

[0341] MALPVTALLLPLALLLHAARP

[0342] SEQ ID NO 14; PRT; Human IL15 (codon optimization)

[0343] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0344] SEQ ID NO 15; PRT; Self-cleaving T2A sequence

[0345] EGRGSLLTCGDVEENPGP

[0346] SEQ ID NO 16; PRT; VHH CDR1 of W3566-FP20R3-1D5-z2

[0347] GSIDSINAIA

[0348] SEQ ID NO 17; PRT; VHH CDR2 of W3566-FP20R3-1D5-z2

[0349] FISSGGFPDYADSVKG

[0350] SEQ ID NO 18; PRT; VHH CDR3 of W3566-FP20R3-1D5-z2

[0351] ERVWGGKIYNY

[0352] SEQ ID NO 19; PRT; VHH CDR1 of W3566-FP20R3-1D5-z2-m17

[0353] GSIDSINAIA

[0354] SEQ ID NO 20; PRT; VHH CDR2 of W3566-FP20R3-1D5-z2-m17

[0355] FISSGGFPDYADWVKG

[0356] SEQ ID NO 21; PRT; VHH CDR3 of W3566-FP20R3-1D5-z2-m17

[0357] EEVWGGKIYNY

[0358] SEQ ID NO 22; PRT; VHH CDR1 of W3566-FP20R3-1D5-z2-m52

[0359] GSIWSINAIA

[0360] SEQ ID NO 23; PRT; VHH CDR2 of W3566-FP20R3-1D5-z2-m52

[0361] FISSGGFPDYADSVKG

[0362] SEQ ID NO 24; PRT; VHH CDR3 of W3566-FP20R3-1D5-z2-m52

[0363] ERVWGGKIYNY

[0364] SEQ ID NO 25; PRT; VHH CDR1 of W306109-P7R2-1G4 and W306109-P7R2-1G4-z02

[0365] GRTRSGADMA

[0366] SEQ ID NO 26; PRT; VHH CDR2 of W306109-P7R2-1G4 and W306109-P7R2-1G4-z02

[0367] AITWNDGLTYYADSVKG

[0368] SEQ ID NO 27; PRT; VHH CDR3 of W306109-P7R2-1G4 and W306109-P7R2-1G4-z02

[0369] RFRARWWD

[0370] SEQ ID NO 28; DNA; W3566-FP20R3-1D5-z2 (anti-BCMA VHH)

[0371] GAAGTTCAGCTGGTTGAAAGCGGCGGTGGTCTGGTTCAGCCGGGTGGTAGTCTGCGTCTGAGTTGCGCCGCGAGTGGCAGCATCGATAGCATCAACGCCATTGCGTGGTACCGTCAAGCCCCGGGCAAACAGCGCGAACTGGTGAGCTTCATCAGCAGCGGCGGCTTTCCGGATTACGCGGATAGCGTTAAAGGCCGCTTCACCATTAGCCGCGACAACAGCAAGAACACCGTTTATCTGCAGATGAACAGTCTGCGCGCCGAAGATACCGCCGTGTACTACTGCAATGCCGAACGCGTTTGGGGTGGCAAGATCTACAACTACTGGGGCCAAGGCACGATGGTTACCGTTAGCAGC

[0372] SEQ ID NO: 29; DNA; W3566-FP20R3-1D5-z2-m17 (anti-BCMA VHH)

[0373] GAAGTGCAGCTGGTTGAAAGCGGCGGTGGTCTGGTTCAGCCGGGTGGTAGTCTGCGTCTGAGTTGCGCCGCGAGCGGTAGCATCGATAGCATCAACGCCATCGCGTGGTATCGTCAAGCCCCGGGCAAACAGCGCGAGCTGGTTAGCTTCATCAGCAGCGGCGGCTTCCCGGATTACGCCGATTGGGTGAAAGGCCGCTTTACCATCAGCCGCGACAACAGCAAGAACACCGTGTATCTGCAGATGAACAGTCTGCGCGCGGAAGATACCGCCGTGTACTACTGCAATGCCGAGGAAGTGTGGGGCGGCAAGATCTACAACTACTGGGGCCAAGGCACGATGGTTACCGTTAGCAGC

[0374] SEQ ID NO 30; DNA; W3566-FP20R3-1D5-z2-m52 (anti-BCMA VHH)

[0375] GAAGTTCAGCTGGTTGAAAGCGGCGGTGGTCTGGTTCAGCCGGGTGGTAGTCTGCGTCTGAGTTGCGCCGCGAGTGGCAGCATCTGGAGCATCAACGCCATTGCGTGGTACCGTCAAGCCCCGGGCAAACAGCGCGAACTGGTGAGCTTCATCAGCAGCGGCGGCTTTCCGGATTACGCGGATAGCGTTAAAGGCCGCTTCACCATTAGCCGCGACAACAGCAAGAACACCGTTTATCTGCAGATGAACAGTCTGCGCGCCGAAGATACCGCCGTGTACTACTGCAATGCCGAACGCGTTTGGGGTGGCAAGATCTACAACTACTGGGGCCAAGGCACGATGGTTACCGTTAGCAGC

[0376] SEQ ID NO 31; DNA; W306109-P7R2-1G4 (anti-GPRC5D VHH)

[0377] CAGGTGCAGCTGGTGGAATCTGGGGGAGGCTTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAATCTCTGGACGCACCCGTAGTGGCGCTGACATGGCCTGGTACCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTCGCAGCGATTACCTGGAATGATGGACTGACATACTATGCAGACTCCGTGAAAGGCCGATTCGCCATCTCCAGAGACGACGCCAAGAACACGCTGCTGACTCTGCAGATGAACAGCCTGAAACCTGAGGACACGGCCATCTATTACTGTAATAATCGGTTTCGTGCTAGGTGGTGGGACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

[0378] SEQ ID NO 32; DNA; W306109-P7R2-1G4-z02 (anti-GPRC5D VHH)

[0379] GAAGTGCAGCTGGTGGAGAGCGGCGGGGGCCTGGTGAAACCCGGCGGGTCCCTGAGACTGAGCTGCGCCGCTAGCGGCAGAACAAGAAGCGGCGCCGACATGGCCTGGTACAGACAAGCCCCCGGCAAGGAGAGAGAGTTCGTGGCCGCCATCACCTGGAACGACGGCCTGACCTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGAGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCCGTGTACTACTGCAACAACAGATTCAGAGCTAGATGGTGGGACTGGGGCCAAGGCACAATGGTGACCGTGAGCAGC

[0380] SEQ ID NO 33; DNA; (G4S)3 linker

[0381] GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCG

[0382] SEQ ID NO 34; DNA; human CD8 signal peptide

[0383] ATGGCCCTGCCCGTGACCGCCCTGCTCCTGCCCCTGGCCCTGCTCCTGCATGCTGCTAGACCC

[0384] SEQ ID NO 35; DNA; human CD8 hinge and transmembrane (TM) domain

[0385] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC

[0386] SEQ ID NO 36; DNA; human 4-1BB-derived intracellular (ICD) signaling domain

[0387] AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[0388] SEQ ID NO 37; DNA; Human CD3ζ-derived intracellular signaling domain

[0389] CGGGTGAAGTTCTCCAGGTCTGCCGACGCCCCCGCCTACAAGCAGGGCCAGAACCAGCTGTACAATGAGCTGAACCTGGGCCGGCGGGAGGAGTATGACGTGCTGGATAAGAGGCGCGGCCGGGACCCCGAGATGGGCGGTAAACCTAGAAGAAAAAATCCACAGGAAGGACTGTATAATGAACTGCAGAAGGATAAAATGGCTGAGGCATATAGTGAAATTGGCATGAAAGGGGAGAGGAGAAGAGGAAAAGGACATGATGGACTGTATCAGGGACTGAGCACAGCTACAAAAGATACATATGATGCCCTGCACATGCAGGCCCTGCCCCCTAGA

[0390] SEQ ID NO 38; DNA; BCMA-1D5, W3566-FP20R3-1D5-z2-m17

[0391] GAGGTGCAGCTGGTGGAATCCGGCGGGGGCCTCGTGCAACCCGGCGGCTCCCTCAGACTGAGCTGCGCCGCTAGCGGCAGCATCGACAGCATCAACGCCATCGCCTGGTACAGACAAGCCCCCGGCAAGCAGAGAGAGCTGGTGAGCTTCATCAGCAGCGGCGGCTTCCCCGACTACGCCGACTGGGTGAAGGGCAGATTCACCATCAGCAGAGACAACAGCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCAACGCCGAAGAGGTGTGGGGCGGCAAGATCTACAACTACTGGGGCCAAGGCACAATGGTGACCGTGAGCAGC

[0392] SEQ ID NO 39; DNA; BCMA-1D5~(G4S)x3 linker~GPRC5D-1G4

[0393] GAAGTGCAGCTGGTGGAGTCCGGCGGGGGGCTGGTGCAGCCCGGCGGCAGCCTGAGACTGTCCTGCGCCGCTAGCGGGAGCATTGACAGCATCAACGCCATCGCTTGGTACAGACAAGCCCCCGGCAAGCAGCGGGAGCTGGTGAGCTTCATCAGCTCCGGGGGGTTCCCCGACTACGCCGACTGGGTGAAGGGCAGATTCACCATCAGCAGAGACAACAGCAAGAACACCGTCTATCTGCAAATGAACTCCCTGAGAGCCGAGGATACCGCCGTGTACTATTGTAACGCCGAAGAGGTGTGGGGCGGCAAGATCTACAACTATTGGGGGCAAGGCACAATGGTGACCGTGAGCAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGAAGTGCAACTCGTGGAGAGCGGCGGGGGCCTGGTGAAGCCTGGCGGCTCCCTGAGACTGAGCTGCGCTGCTAGCGGCAGAACAAGAAGCGGCGCCGACATGGCTTGGTATCGGCAAGCCCCCGGCAAGGAGCGGGAGTTCGTGGCCGCCATCACCTGGAACGACGGCCTGACCTACTACGCCGACAGCGTGAAAGGCAGATTCACAATCAGCAGAGACGATTCCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAGCCCGAGGACACAGCTGTGTACTACTGCAACAACAGATTCAGAGCTAGATGGTGGGATTGGGGCCAAGGCACAATGGTGACCGTCAGCAGC

[0394] SEQ ID NO 40; DNA; Human CD8a signal peptide of IL15

[0395] ATGGCCCTGCCCGTGACCGCCCTGCTCCTGCCCCTGGCCCTGCTGCTCCATGCCGCTAGACCC

[0396] SEQ ID NO 41; DNA; Human IL15 (codon-optimized)

[0397] AACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATTCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCTAGCTGCAAGGTGACCGCCATGAAGTGCTTCCTGCTGGAGCTGCAAGTGATCAGCCTGGAGAGCGGCGACGCTAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACAAGC

[0398] SEQ ID NO 42; DNA; self-cleaving T2A sequence

[0399] GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT

Claims

1. A bispecific chimeric antigen receptor (CAR) comprising: (a) B cell maturation antigen (BCMA) targeting domain; and (b) Targeting domain of G protein-coupled receptor C family 5 member D (GPRC5D).

2. The CAR as claimed in claim 1, wherein, The BCMA targeting domain and the GPRC5D targeting domain: (i) Independently selected from or derived from antibodies or their antigen-binding fragments; and / or (ii) Independently selected from or derived from single-chain variable fragments (scFv), heavy chain variable domains (VHH) of heavy chain antibodies, and nanobodies; and / or (iii) Connected, merged or coupled to each other in any order, optionally with or without joints.

3. The CAR as described in claim 1, wherein, The BCMA targeting domain comprises: VH or VHH CDR1 selected from SEQ ID NO:16, 19 and 22; VH or VHH CDR2 selected from SEQ ID NO:17, 20 and 23; and VH or VHH CDR3 selected from SEQ ID NO:18, 21 and 24; and / or The BCMA targeting domain includes VH or VHH CDR1-3, which are SEQ ID NO: 16, 17 and 18; SEQ ID NO: 19, 20 and 21; or SEQ ID NO: 22, 23 and 24; and / or The BCMA targeting domain is described below: (a1) Contains a polypeptide having an amino acid sequence SEQ ID NO: 1, 2, 3 or 11; and / or (a2) Contains a polypeptide that has greater than 80% sequence identity with SEQ ID NO: 1, 2, 3 or 11 and has binding specificity to BCMA; and / or (a3) Encoded by a nucleic acid molecule having a nucleotide sequence SEQ ID NO: 28, 29, 30 or 38; and / or (a4) Encoded by a nucleic acid molecule that has a sequence identity greater than 80% with the nucleotide sequence SEQ ID NO: 28, 29, 30 or 38 and encodes a BCMA-binding polypeptide.

4. The CAR as claimed in claim 1, wherein, The GPRC5D targeting domain contains VH or VHH CDR1-3, which are SEQ ID NO: 25, 26 and 27, respectively; and / or The GPRC5D targeting domain is described below: (b1) Contains a polypeptide having an amino acid sequence SEQ ID NO: 4 or 5; and / or (b2) Contains a polypeptide that has greater than 80% sequence identity with SEQ ID NO: 4 or 5 and has binding specificity to GPRC5D; and / or (b3) Encoded by a nucleic acid molecule having a nucleotide sequence SEQ ID NO: 31 or 32; and / or (b4) Encoded by a nucleic acid molecule that has a sequence identity greater than 80% with the nucleotide sequence SEQ ID NO: 31 or 32 and encodes a GPRC5D binding polypeptide.

5. The CAR as claimed in claim 1, wherein, The CAR includes a BCMA target domain and a GPRC5D target domain connected in series in any order; and / or The CAR contains a polypeptide having the amino acid sequence SEQ ID NO: 12; and / or The CAR comprises a polypeptide that has greater than 80% sequence identity with SEQ ID NO: 12 and is capable of binding to BCMA and GPRC5D; and / or The CAR comprises a polypeptide encoded by a nucleic acid molecule having the nucleotide sequence SEQ ID NO: 39; and / or The CAR contains a polypeptide encoded by a nucleic acid molecule that has greater than 80% sequence identity with the nucleotide sequence SEQ ID NO: 39 and encodes a polypeptide capable of binding to BCMA and GPRC5D.

6. The CAR as claimed in claim 1, wherein, The CAR also includes one or more domains or units selected from the following group: (c) Extracellular signaling domains; (d) Extracellular hinge domain; (e) Transmembrane (TM) domain; (f) One or more intracellular signal transduction domains (ICDs); (g) Armored units; and (h) One or more independently selected joints, which are located between two domains or between a domain and a unit.

7. The CAR of claim 6, wherein (c) The extracellular signaling domain is a CD8 signal peptide; and / or (d) The extracellular hinge domain is a CD8 hinge or an IgG4Fc hinge; and / or (e) The transmembrane (TM) domain is selected from the group consisting of: CD8 TM, CD4 TM, CD28 TM, CD16 TM, EPOR TM, CD3ζ TM; and / or (f) The intracellular signal transduction domain (ICD) is selected from the following group: 4-1BB (CD137), CD3ζ intracellular domain, CD8α, CD8β, co-stimulatory signal transduction domain, selected from CD27, CD28, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands, specifically binds to CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, IT GAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 ( Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162); and / or (h) The joint is independently selected from flexible joints, rigid joints, and detachable joints; and / or The connector is independently selected from serine-glycine connectors, glycine connectors, and (EAAAK). n Connectors (n is 1, 2, 3, 4, 5, 6, 7, 8, or 9), T2A connectors; and / or The connector is (G4S). n Connector, n is 1, 2, 3, 4, 5, 6, 7, 8 or 9.

8. The CAR as claimed in claim 1, wherein, The CAR is an armored CAR, which contains one or more armored units; and / or The armored unit includes a connector, a signal structure domain, and an armor structure domain located between the CAR and the armored unit; and / or Wherein, the joint is a self-destructing joint; and / or The connectors are T2A, E2A, F2A, P2A, BmCPV2A, and BmIFV2A connectors; and / or Wherein, the armor structure domain is selected from IL15, IL12, IL18, IL7, IL4, CXCL9, CXCL10 or any combination thereof; and / or The armor unit comprises a combination of a self-cleaving T2A connector, an IL15 CD8a signal peptide, and IL15.

9. The CAR as claimed in claim 1, wherein, The CAR includes (a') and (b') or (ab'): (a') the BCMA targeting domain as defined in claim 4; and (b') The GPRC5D targeting domain as defined in claim 5; or (ab') the cascaded BCMA targeting domain and GPRC5D targeting domain as defined in claim 6; and The CAR mentioned above also includes: (c') The CD8 signal peptide in the extracellular signaling domain; (d')CD8TM structural domain; (e') CD8 TM in the TM structure domain; (f') the 4-1BB derived ICD and / or CD3ζ derived ICD in the ICD structural domain; (g') IL-15, the T2A autolytic linker, and the CD8a signal peptide of IL-15 in the armored unit; and (h') is a (G4S)3 connector located between (a') and (b') or in (ab').

10. The CAR as claimed in claim 1, wherein, The CAR includes (I) Possesses a combination of domains of the amino acid sequence SEQ ID NO: 7, 12, 8, 9, 10; or (II) Having a combination of domains of the amino acid sequence SEQ ID NO: 7, 12, 8, 9, 10, 13, 14 and 15; or (III) Combinations of domains that have a sequence identity greater than 80% with the combinations defined in (I) or (I) and are capable of combining with BCMA and GPRC5D.

11. An anti-GPRC5D antibody or an antigen-binding fragment thereof, comprising a GPRC5D binding domain, wherein the GPRC5D binding domain comprises heavy chain (VH) complementarity-determining regions (CDRs) 1, 2, and 3, and wherein the VH CDR1 has the amino acid sequence of SEQ ID NO: 25, the VH CDR2 has the amino acid sequence of SEQ ID NO: 26, and the VH CDR3 has the amino acid sequence of SEQ ID NO:

27.

12. The antibody or antigen-binding fragment as described in claim 11, in, The GPRC5D binding fragment comprises a polypeptide having the amino acid sequence SEQ ID NO: 4 or 5; and / or The GPRC5D binding fragment comprises a polypeptide with greater than 80% sequence identity to SEQ ID NO: 4 or 5 and binding specificity to GPRC5D; and / or Wherein, the GPRC5D binding fragment is encoded by a nucleic acid molecule having the nucleotide sequence SEQ ID NO: 31 or 32; and / or Wherein, the GPRC5D binding fragment is encoded by a nucleic acid molecule, wherein the nucleic acid molecule has a sequence identity greater than 80% with the nucleotide sequence of SEQ ID NO: 31 or 32 and encodes a GPRC5D binding polypeptide; and / or The antibody or antigen-binding fragment is used to construct the CAR.

13. The antibody or antigen-binding fragment as described in claim 11, wherein, The antibody or antigen-binding fragment is a monovalent or multivalent monoclonal antibody, nanobody, scFv, Fab, F(ab)2, Fv; and / or Wherein, the antibody is a humanized antibody, a chimeric antibody; and / or The antibody or antigen-binding fragment is used to construct modified NK cells containing one or more CARs.

14. An anti-BCMA antibody or an antigen-binding fragment thereof, comprising a BCMA-binding domain, wherein the BCMA-binding domain comprises heavy chain (VH) complementarity-determining regions (CDRs) 1, 2, and 3, and wherein the VH CDR1 has an amino acid sequence selected from SEQ ID NO: 16, 19, and 22, the VH CDR2 has an amino acid sequence selected from SEQ ID NO: 17, 20, and 23, and the VH CDR3 has an amino acid sequence selected from SEQ ID NO: 18, 21, and 24.

15. The antibody or antigen-binding fragment as described in claim 14, wherein, The binding domains include VH CDR1-3 selected from the group consisting of: SEQ ID NO: 16, 17 and 18; SEQ ID NO: 19, 20 and 21; or SEQ ID NO: 22, 23 and 24.

16. The antibody or antigen-binding fragment of claim 14, wherein... in, The BCMA-binding fragment comprises a polypeptide having the amino acid sequence SEQ ID NO: 1, 2, 3, or 11; and / or Wherein, the BCMA-binding fragment comprises a polypeptide with greater than 80% sequence identity to SEQ ID NO: 1, 2, 3 or 11 and binding specificity to BCMA; and / or Wherein, the BCMA-binding fragment is encoded by a nucleic acid molecule having the nucleotide sequence SEQ ID NO: 28, 29, 30 or 38; and / or Wherein, the BCMA-binding fragment is encoded by a nucleic acid molecule, wherein the nucleic acid molecule has a sequence identity greater than 80% with the nucleotide sequence of SEQ ID NO: 28, 29, 30 or 38 and encodes a BCMA-binding polypeptide; and / or The antibody or antigen-binding fragment is used to construct the CAR.

17. The antibody or antigen-binding fragment as described in claim 14, wherein, The antibody or antigen-binding fragment is a monovalent or multivalent monoclonal antibody, nanobody, scFv, Fab, F(ab)2, Fv; and / or Wherein, the antibody is a humanized antibody, a chimeric antibody; and / or The antibody or antigen-binding fragment is used to construct modified NK cells containing one or more CARs.

18. One or more polynucleotide molecules encoding a CAR according to any one of claims 1-10 or an antibody or antigen-binding fragment according to any one of claims 11-17.

19. One or more vectors comprising a CAR encoding a polynucleotide molecule according to any one of claims 1-10, an antibody or antigen-binding fragment according to any one of claims 11-17, or a polynucleotide molecule according to claim 18.

20. A modified NK cell comprising one or more CARs according to any one of claims 1-10, one or more CARs constructed from an antibody or antigen-binding fragment according to any one of claims 11-17, one or more polynucleotide molecules encoding a CAR according to claim 18, or transduced using the vector according to claim 19.

21. The modified NK cells of claim 20, wherein, The NK cells are derived from the following groups: umbilical cord blood, peripheral blood, and / or placenta of vertebrates (e.g., human or rodent cells), and induced pluripotent stem cells (iPSCs); and / or Wherein, the NK cells are autologous homologous cells or allogeneic cells; and / or Wherein, the NK cells are transduced by the same or different vectors; and / or The vector is selected from the group consisting of: plasmids, viruses, bacteria, bacteriophages; and / or The modified NK cells are present in cell populations, cell cultures, or products.

22. A product comprising one or more CARs selected from any one of claims 1-10, an antibody or antigen-binding fragment selected from any one of claims 11-17, one or more polynucleotide molecules selected from claim 18, one or more vectors selected from claim 19, and one or more modified NK cells selected from any one of claims 20-21.

23. A method for preparing the modified NK cells according to any one of claims 20-21, comprising the following steps: (i) Modifying NK cells to include an armored or unarmored chimeric antigen receptor (CAR) as claimed in any one of claims 1-10 or a CAR constructed from an antibody or antigen-binding fragment as claimed in any one of claims 11-17; and (ii) optionally, expanding and collecting the modified NK cells containing the CAR.

24. Use of the modified NK cells as described in any one of claims 20-21 in the preparation of a product for the treatment of multiple myeloma (MM).

25. A method for treating multiple myeloma (MM) in a subject with this need, comprising administering an effective amount of the modified NK cells as described in any one of claims 20-21.

26. The modified NK cells according to any one of claims 20-21, for the treatment of multiple myeloma (MM).

27. The use as described in claim 24, or the method as described in claim 25, or the modified NK cells for treating multiple myeloma as described in claim 26, wherein said treatment is adoptive cell therapy, particularly CAR-NK adoptive cell therapy; and / or in, The MM is responsive MM, non-responsive MM, progressive MM, relapsed MM, and / or refractory MM; and / or Wherein, the MM is an RRMM; and / or The MM is classified as stage I, II, or III MM according to the International Myeloma Staging System.

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